Bioconjugates of cyanine dyes

ABSTRACT

Compounds are disclosed that are useful for noninvasive imaging in the near-infrared spectral range. Bioconjugate cyanine compounds of Formula II are presented: 
     
       
         
         
             
             
         
       
     
     wherein Q is a portion of a polymethine bridge selected from the group consisting of:

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. Non-Provisional applicationSer. No. 13/831,345 (filed Mar. 14, 2013), which is a continuation ofthe International PCT Application No. PCT/US2011/057134 (filed Oct. 20,2011), which claims the benefit of U.S. Provisional Patent ApplicationNos. 61/405,158 and 61/405,161 (both filed Oct. 20, 2010), which arehereby incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

Cyanine dyes have been widely used for labeling ligands or biomoleculesfor a variety of applications such as DNA sequencing. (See, for example,U.S. Pat. No. 5,571,388 for exemplary methods of identifying strands ofDNA by means of cyanine dyes.) More recently, they have been used foroptical imaging of dye-labeled biomolecules, either in vivo or in vitro.(See, for example, U.S. Pat. No. 7,597,878.) Scientists favor usingcyanine dyes in biological applications because, among other reasons,many of these dyes fluoresce in the near-infrared (NIR) region of thespectrum (600-1000 nm). This makes cyanine dyes less susceptible tointerference from autofluorescence of biomolecules.

Other advantages of cyanine dyes include, for example: 1) cyanine dyesstrongly absorb and fluoresce light; 2) many cyanine dyes do not rapidlybleach under a fluorescence microscope; 3) cyanine dye derivatives canbe made that are effective coupling reagents; 4) many structures andsynthetic procedures are available, and the class of dyes is versatile;and 5) cyanine dyes are relatively small (a typical molecular weight isabout 1,000 daltons), so they do not cause appreciable stericinterference in a way that might reduce the ability of a labeledbiomolecule to reach its binding site or carry out its function.

Despite their advantages, many of the known cyanine dyes have a numberof disadvantages. Some known cyanine dyes are not stable in the presenceof certain reagents that are commonly found in bioassays. Such reagentsinclude ammonium hydroxide, dithiothreitol (DTT), primary and secondaryamines, and ammonium persulfate (APS). Further, some known cyanine dyeslack the thermal stability and photostability that is necessary forbiological applications such as DNA sequencing and genotyping.

For these reasons, stable cyanine dyes are needed for use in labelingbiomolecules as well as in vivo imaging for the diagnosis and prognosisof diseases such as cancer. Such compositions and methods would aid inthe analysis of responses to various therapies. The present inventionsatisfies these and other needs.

BRIEF SUMMARY OF THE INVENTION

Various embodiments of the present invention provide compounds,bioconjugates, methods of labeling, and methods of measuring ordetecting target molecules non-invasively, thus solving the problems ofthe above-described art.

As such, in one embodiment, the present invention provides a compound ofFormula I:

wherein Q is a portion of a polymethine bridge selected from the groupof:

wherein Q is the central portion of either a five- or aseven-polymethine-carbon polymethine bridge;

each R¹ is a member independently selected from the group of -L-Y—Z andalkyl that is additionally substituted with from 0 to 1 R¹³ and from 0to 1 R¹⁶; wherein the alkyl is optionally interrupted by at least oneheteroatom;

each R^(2a) and R^(2b) is a member independently selected from the groupconsisting of alkyl, alkenyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl,amidoalkyl, alkylthioalkyl, carboxyalkyl, alkoxycarbonylalkyl, orsulfonatoalkyl; wherein a carbon of the member is additionallysubstituted with from 0 to 1 R¹⁶; or, alternatively, a R^(2a) and R^(2b)pair, together with the ring carbon to which the R^(2a) and R^(2b) arebonded, join to form a spirocycloalkyl ring, wherein the spirocycloalkylring is additionally substituted with from 0 to 6 R¹⁴ and from 0 to 1R¹⁶, or an exocyclic alkene, wherein the exocyclic alkene isadditionally substituted with from 0 to 2 R¹⁴ and from 0 to 1 R¹⁶;

each R³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and R^(6b) is a memberindependently selected from the group consisting of hydrogen, alkyl,alkenyl, halo, hydroxyl, alkoxy, amino, cyano, carboxyl, alkoxycarbonyl,amido, sulfonato, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, andsulfonatoalkyl; wherein a carbon of the member is additionallysubstituted with from 0 to 1 R¹⁶; or, alternatively, a pair of saidmembers that is selected from the group consisting of R³ and R^(4a), anR^(4a) and R^(5a), and an R^(5a) and R^(6a), together with the pair ofatoms to which the pair of said members is bonded, joins to form an arylring, wherein the aryl ring is additionally substituted with from 0 to 2R¹⁴ and from 0 to 1 R¹⁶;

each R⁷ is a member independently selected from the group consisting ofhydrogen and alkyl; wherein the alkyl is additionally substituted withfrom 0 to 3 R¹⁴ and from 0 to 1 R¹⁶; or, alternatively, both R⁷,together with the intervening segment of the polyene to which both R⁷are bonded, join to form a ring, wherein said ring is selected from thegroup consisting of a cycloalkyl and a heterocyclyl ring; wherein thering is additionally substituted with from 0 to 3 R¹⁴ and from 0 to 1R¹⁶;

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each a member independently selected fromthe group consisting of hydrogen, alkyl, alkenyl, halo, alkoxy,sulfonato, hydroxyl, amino, carboxyl, alkoxycarbonyl, cyano, amido,thioacetyl, and -L-Y—Z; wherein, if present, at least one memberselected from the group consisting of R⁸, R⁹, and R¹⁰ is -L-Y—Z;

each R¹³ is a member independently selected from the group consisting ofhydroxyl, amino, carboxyl, alkoxycarbonyl, cyano, amido, sulfonato, andthioacetyl;

each R¹⁴ is a member independently selected from the group consisting ofalkyl, alkenyl, halo, hydroxyl, alkoxy, amino, cyano, carboxyl,alkoxycarbonyl, amido, sulfonato, alkoxycarbonylalkyl, and alkoxyalkyl;wherein the alkyl or alkenyl is additionally substituted with from 0 to1 R¹³ and from 0 to 1 R¹⁶;

each L is an optional member independently selected from the groupconsisting of a bond, a C₁-C₂₀ alkylene, and a C₁-C₂₀ alkenylene;wherein the alkylene or alkenylene is optionally interrupted by at leastone heteroatom;

each Y is an optional member independently selected from the groupconsisting of a bond, —O—, —S—, —NH—, —NHC(O)—, —C(O)NH—, —NR¹⁵—,—NR¹⁵C(O)—, —C(O)NR¹⁵—, —N(Z)—, —N(Z)C(O)—, and —C(O)N(Z)—;

each Z is independently selected from the group consisting of -L-R¹³ and-L-R¹⁶, or in an alternative embodiment, —Y—Z is a member selected fromthe group of —N(Z)₂, —N(Z)C(O)Z, and —C(O)N(Z)₂, wherein the two Zgroups may optionally be linked to form a cycloalkynyl group;

each R¹⁵ is a member independently selected from the group consisting ofalkyl and alkoxycarbonylalkyl; wherein the alkyl is optionallyinterrupted by at least one heteroatom;

each R¹⁶ is independently a member selected from the group consisting ofactivated acyl, acrylamido, optionally substituted alkylsulfonate ester,azido, optionally substituted arylsulfonate ester, optionallysubstituted amino, aziridino, boronato, cycloalkynyl,cycloalkynylcarbonyl, diazo, formyl, glycidyl, halo, haloacetamidyl,haloalkyl, haloplatinato, halotriazino, hydrazinyl, imido ester,isocyanato, isothiocyanato, maleimidyl, mercapto, phosphoramidityl, aphotoactivatable moiety, vinyl sulfonyl, alkynyl, cycloalkynyl,spirocycloalkynyl, a pegylated azido, a pegylated alkynyl, a pegylatedcycloalkynyl, a pegylated spirocycloalkynyl, an ortho substitutedphosphinyl aryl ester (e.g., TPPME), and an ortho substituted phosphineoxide aryl ester; and wherein said compound has a balanced charge.

In another embodiment, the present invention provides a bioconjugate ofthe Formula II:

wherein Q^(L) is a portion of a polymethine bridge selected from thegroup consisting of:

wherein Q^(L) is the central portion of either a five- or aseven-polymethine-carbon polymethine bridge;

wherein R¹, R^(2a), R^(2b), R³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a),R^(6b), R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²R¹³, R¹⁴, R¹⁵, R¹⁶, L, and Y are aspreviously defined for the compound of Formula I;

each Z is independently selected from the group consisting of -L-R¹³ and-L-R^(L);

each R^(L) comprises a linking group and a biomolecule connectedthereto, wherein the compound comprises at least one R^(L), and whereinthe compound has a balanced charge.

In yet another embodiment, the present invention provides a method orprocess for labeling a ligand or biomolecule with a compound of FormulaI, the method comprising contacting a ligand or biomolecule with acompound having Formula Ito generate the corresponding bioconjugatecompound of Formula II.

In still yet another embodiment, the compounds of Formula I or II can beused as in vitro or in vivo optical imaging agents of tissues and organsin various biomedical applications. In one aspect, the present inventionprovides a method for imaging, the method comprising administering acompound of Formula I or Formula II.

Further aspects, objects, and advantages of the invention will becomeapparent upon consideration of the detailed description and figures thatfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the UV absorption and emission curves for compound10.

FIG. 2 illustrates a Western blot total fluorescence comparison of p38GAR antibody conjugates with compound 10, compound 20, and IRDye® 680(“LICOR 680”).

FIG. 3 shows a series of images from two tumor-bearing mice afterintravenously receiving 1 nmol of an 10/cyclo(RGDfK) peptide conjugate.The tumors were visible by 5 h post-injection.

FIG. 4 shows the tumors from the two mice of FIG. 2. After the tumors'removal, they were imaged on the Pearl Impulse and weighed.

FIG. 5 illustrates a Western blot total fluorescence comparison of threeGAM antibody conjugates with 10 at different D/P ratios. GAM antibodyconjugates with 20, IRDye® 680, and AlexaFluor® 680 were used ascontrols. (A) 10; D/P 1.47. (B) 10; D/P 2.60. (C) 10; D/P 4.42. (D) 20;D/P 2. 16. (E) IRDye® 680; D/P 3.13. (F) AlexaFluor® 680; D/P 4.

FIG. 6A shows the 10's integrated intensity values (local backgroundsubtracted) for each of the conjugates of FIG. 4. FIG. 6B provides themean local background for each conjugate. The error bars representstandard deviation for six background sets.

FIG. 7 shows Pearl® Impulse images of nude mice taken 24 hpost-injection with a conjugate of 10 and a tetracycline antibiotic.FIG. 7A shows a mouse in the top panels received 4 nmol intravenously(IV); FIG. 7B shows the mouse in the bottom panels received 4 nmolintraperitoneally (IP).

FIG. 8 shows a Pearl Impulse image of a nude mouse taken approximately10 min after intradermal (ID) injection of an 10/hyaluronin conjugate(10-HA). This animal was imaged over time and 96 h images are presentedin FIG. 9.

FIG. 9 shows Pearl Impulse images of nude mice taken approximately 96 hafter ID injection of 10-HA. The images include (A) ventral and (B)dorsal views of the mouse prior to sacrifice and (C), (D), (E), and (F)are dissection panels.

FIG. 10 shows Pearl Impulse images of nude mice taken approximately 24 hpost IV injection of 10-HA. The white arrow points to tumor locations onthe dorsal view.

FIG. 11 shows Pearl Impulse images of two nude mice taken approximately1 min post injection of 1 nmol 10/polyethylene glycol (PEG) conjugate.The mice had A431 tumors on their right hips.

FIG. 12 shows Pearl Impulse images of two nude mice taken approximately24 h post-injection of 1 nmol 10/PEG conjugate. The white arrow pointsto a tumor.

FIGS. 13A and B illustrate compound 20's absorbance and emission maximain methanol (680 nm; Panel A); and in phosphate-buffered saline solution(676 nm; Panel B).

FIG. 14 illustrates the absorbance of compound 20-labeled GAM antibodyin 1:1 PBS:methanol.

FIG. 15 illustrates the absorbance of compound 20-labeled lactalbumin in1:1 phosphate-buffered saline (PBS):methanol.

FIG. 16 illustrates a comparison of fluorescence and D/P for compound20-labeled goat anti-rabbit (GAR) antibody.

FIG. 17 illustrates a comparison of fluorescence and D/P for AlexaFluor®680-labeled goat anti-rabbit (GAR) antibody.

FIG. 18 illustrates a comparison of relative fluorescence betweencompound 20 and AlexaFluor® 680.

FIG. 19A-B illustrate a comparison of photostability among IRDye® 700DX,compound 20, and AlexaFluor® 680 at 50 fmoles (Panel A); and 25 fmoles(Panel B).

FIG. 20A-B illustrate a comparison of cell staining and relativefluorescence between compound 20 (Panel A); and AlexaFluor® 680 (PanelC). FIG. 10B and FIG. 10C compare fluorescence intensities,respectively.

FIG. 21A-C illustrate the immunofluorescence staining of HER2 proteinwith compound 20-labeled GAR antibodies. Cells were incubated withrabbit anti-HER2 mAb, followed by 20-labeled GAR secondary antibody(Panel A). Sytox green was used to stain the nuclei (Panel B); a mergedimage is illustrated in Panel C.

FIG. 22A-C illustrate a Western blot total fluorescence comparison ofβ-actin GAM antibody conjugates with compound 20 (Panel A); IRDye® 680(Panel B); and AlexaFluor® 680 (Panel C).

FIG. 23 illustrates the response of the β-actin GAM antibody conjugates'fluorescence intensity at increasing concentrations of cell lysate.

FIG. 24A-C illustrates a Western blot total fluorescence comparison ofp38 GAR antibody conjugates with compound 20, IRDye® 680, andAlexaFluor® 680.

FIG. 25A-C illustrate a two-color Western blot total fluorescencecomparison of Akt GAM antibody conjugates with IRDye® 680 (Panel A);compound 20 (Panel B); and AlexaFluor® 680 (Panel C). Rabbit mAb (actin)was detected with an IRDye® 800CW/GAR antibody conjugate.

FIG. 26A-B illustrate a Western blot total fluorescence comparison ofAkt GAR antibody conjugates with compound 20, IRDye® 680, andAlexaFluor® 680 in Panel A. A control experiment without primaryantibody is illustrated in Panel B.

FIG. 27 illustrates the relative fluorescent intensity of compound 21and compound 10 with increasing concentration.

FIG. 28A-B illustrate in Panel A total intensity as a function of timepost-injection of the Compound 10's clearance from various injectionsites of an imaged mammal; Panel B shows total intensity as a functionof time post-injection of Compound 10 clearance from various injectionsites of an imaged mammal.

FIG. 29 illustrates an imaged mammal with compounds of the presentinvention.

FIG. 30A-C illustrate in Panel A a plate based assay with compound 10;Panel B shows relative intensity of compound 10 binding in A431 cells;Panel C shows competitive binding of labeled and unlabeled EpidermalGrowth Factor (EGF).

FIG. 31 illustrates in Panel A in vivo imaging of compound 10 conjugatebinding in a mammal at various doses, 2 nmol is a preferredconcentration; Panel B shows in vivo imaging of compound 10 conjugatebinding in a mammal at various doses (e.g., 2 nmol).

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forexample, an embodiment of a method of imaging that comprises using acompound set forth in claim 1 would include an aspect in which themethod comprises using two or more compounds set forth in claim 1.

The term “about” as used herein to modify a numerical value indicates adefined range around that value. If “X” were the value, “about X” wouldindicate a value from 0.9X to 1.1X, and more preferably, a value from0.95X to 1.05X. Any reference to “about X” specifically indicates atleast the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X,1.03X, 1.04X, and 1.05X. Thus, “about X” is intended to teach andprovide written description support for a claim limitation of, e.g.,“0.98X.”

When the quantity “X” only allows whole-integer values (e.g., “Xcarbons”) and X is at most 15, “about X” indicates from (X−1) to (X+1).In this case, “about X” as used herein specifically indicates at leastthe values X, X−1, and X+1. If X is at least 16, the values of 0.90X and1.10X are rounded to the nearest whole-integer values to define theboundaries of the range.

When the modifier “about” is applied to describe the beginning of anumerical range, it applies to both ends of the range. Thus, “from about700 to 850 nm” is equivalent to “from about 700 nm to about 850 nm.”When “about” is applied to describe the first value of a set of values,it applies to all values in that set. Thus, “about 680, 700, or 750 nm”is equivalent to “about 680 nm, about 700 nm, or about 750 nm.” However,when the modifier “about” is applied to describe only the end of therange or only a later value in the set of values, it applies only tothat value or that end of the range. Thus, the range “about 2 to about10” is the same as “about 2 to about 10,” but the range “2 to about 10”is not.

“Activated acyl” as used herein includes a —C(O)-LG group. “Leavinggroup” or “LG” is a group that is susceptible to displacement by anucleophilic acyl substitution (i.e., a nucleophilic addition to thecarbonyl of —C(O)-LG, followed by elimination of the leaving group).Representative leaving groups include halo, cyano, azido, carboxylicacid derivatives such as t-butylcarboxy, and carbonate derivatives suchas i-BuOC(O)O—. An activated acyl group may also be an activated esteras defined herein or a carboxylic acid activated by a carbodiimide toform an anhydride or mixed anhydride —OC(O)R^(a) or —OC(NR^(a))NHR^(b),wherein R^(a) and R^(b) are members independently selected from thegroup consisting of C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, C₁-C₆ alkoxy,cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl. Preferredactivated acyl groups include activated esters.

“Activated ester” as used herein includes a derivative of a carboxylgroup that is more susceptible to displacement by nucleophilic additionand elimination than an ethyl ester group (e.g., an NHS ester, asulfo-NHS ester, a PAM ester, or a halophenyl ester). Representativecarbonyl substituents of activated esters include succinimidyloxy(—OC₄H₄NO₂), sulfosuccinimidyloxy (—OC₄H₃NO₂SO₃H), -1-oxybenzotriazolyl(—OC₆H₄N₃); 4-sulfo-2,3,5,6-tetrafluorophenyl; or an aryloxy group thatis optionally substituted one or more times by electron-withdrawingsubstituents such as nitro, fluoro, chloro, cyano, trifluoromethyl, orcombinations thereof (e.g., pentafluorophenyloxy). Preferred activatedesters include succinimidyloxy and sulfosuccinimidyloxy esters.

“Acyl” as used herein includes an alkanoyl, aroyl, heterocycloyl, orheteroaroyl group as defined herein. Representative acyl groups includeacetyl, benzoyl, nicotinoyl, and the like.

“Alkanoyl” as used herein includes an alkyl-C(O)— group wherein thealkyl group is as defined herein. Representative alkanoyl groups includeacetyl, ethanoyl, and the like.

“Alkenyl” as used herein includes a straight or branched aliphatichydrocarbon group of 2 to about 15 carbon atoms that contains at leastone carbon-carbon double bond. Preferred alkenyl groups have 2 to about12 carbon atoms. More preferred alkenyl groups contain 2 to about 6carbon atoms. “Lower alkenyl” as used herein includes alkenyl of 2 toabout 6 carbon atoms. Representative alkenyl groups include vinyl,allyl, n-butenyl, 2-butenyl, 3-methylbutenyl, n-pentenyl, heptenyl,octenyl, decenyl, and the like.

“Alkenylene” as used herein includes a straight or branched bivalenthydrocarbon chain containing at least one carbon-carbon double or triplebond. Preferred alkenylene groups include from 2 to about 12 carbons inthe chain, and more preferred alkenylene groups include from 2 to 6carbons in the chain. In one aspect, hydrocarbon groups that contain acarbon-carbon double bond are preferred. In a second aspect, hydrocarbongroups that contain a carbon-carbon triple bond are preferred.Representative alkenylene groups include —CH═CH—, —CH₂—CH═CH—,—C(CH₃)═CH—, —CH₂CH═CHCH₂—, ethynylene, propynylene, n-butynylene, andthe like.

“Alkoxy” as used herein includes an alkyl-O— group wherein the alkylgroup is as defined herein. Representative alkoxy groups includemethoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, heptoxy, and the like.

“Alkoxyalkyl” as used herein includes an alkyl-O-alkylene- group whereinalkyl and alkylene are as defined herein. Representative alkoxyalkylgroups include methoxyethyl, ethoxymethyl, n-butoxymethyl andcyclopentylmethyloxyethyl.

“Alkoxycarbonyl” as used herein includes an ester group; i.e., analkyl-O—CO— group wherein alkyl is as defined herein. Representativealkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,t-butyloxycarbonyl, and the like.

“Alkoxycarbonylalkyl” as used herein includes an alkyl-O—CO-alkylene-group wherein alkyl and alkylene are as defined herein. Representativealkoxycarbonylalkyl include methoxycarbonylmethyl, ethoxycarbonylmethyl,methoxycarbonylethyl, and the like.

“Alkyl” as used herein includes an aliphatic hydrocarbon group, whichmay be straight or branched-chain, having about 1 to about 20 carbonatoms in the chain. Preferred alkyl groups have 1 to about 12 carbonatoms in the chain. More preferred alkyl groups have 1 to 10 or 1 to 6carbon atoms in the chain. “Branched-chain” as used herein includes thatone or more lower alkyl groups such as methyl, ethyl or propyl areattached to a linear alkyl chain. “Lower alkyl” as used herein includes1 to about 6 carbon atoms, preferably 5 or 6 carbon atoms in the chain,which may be straight or branched. Representative alkyl groups includemethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and3-pentyl.

“Alkylene” as used herein includes a straight or branched bivalenthydrocarbon chain of 1 to about 6 carbon atoms. Preferred alkylenegroups are the lower alkylene groups having 1 to about 4 carbon atoms.Representative alkylene groups include methylene, ethylene, and thelike.

“Alkylsulfonate ester” as used herein includes an alkyl-SO₃— groupwherein the alkyl group is as defined herein. Preferred alkylsulfonateester groups are those wherein the alkyl group is lower alkyl.Representative alkylsulfonate ester groups include mesylate ester (i.e.,methylsulfonate ester).

An “optionally substituted” alkylsulfonate ester includes analkylsulfonate ester as defined herein, wherein the aryl group isadditionally substituted with from 0 to 3 halo, alkyl, aryl, haloalkyl,or haloaryl groups as defined herein. Preferred optionally substitutedalkylsulfonate groups include triflate ester (i.e.,trifluoromethylsulfonate ester).

“Alkylthio” as used herein includes an alkyl-S— group wherein the alkylgroup is as defined herein. Preferred alkylthio groups are those whereinthe alkyl group is lower alkyl. Representative alkylthio groups includemethylthio, ethylthio, isopropylthio, heptylthio, and the like.

“Alkylthioalkyl” as used herein includes an alkylthio-alkylene- groupwherein alkylthio and alkylene are defined herein. Representativealkylthioalkyl groups include methylthiomethyl, ethylthiopropyl,isopropylthioethyl, and the like.

“Alkynyl” as used herein includes a straight or branched aliphatichydrocarbon group of 2 to about 15 carbon atoms that contains at leastone carbon-carbon triple bond. Preferred alkynyl groups have 2 to about12 carbon atoms. More preferred alkynyl groups contain 2 to about 6carbon atoms. “Lower alkynyl” as used herein includes alkynyl of 2 toabout 6 carbon atoms. Representative alkynyl groups include propynyl,2-butynyl, 3-methylbutynyl, n-pentynyl, heptynyl, and the like.

“Amido” as used herein includes a group of formula Y₁Y₂N—C(O)— whereinY₁ and Y₂ are independently hydrogen, alkyl, or alkenyl; or Y₁ and Y₂,together with the nitrogen through which Y₁ and Y₂ are linked, join toform a 4- to 7-membered azaheterocyclyl group (e.g., piperidinyl).Representative amido groups include primary amido (H₂N—C(O)—),methylamido, dimethylamido, diethylamido, and the like. Preferably,“amido” is an —C(O)NRR′ group where R and R′ are members independentlyselected from the group consisting of H and alkyl. More preferably, atleast one of R and R′ is H.

“Amidoalkyl” as used herein includes an amido-alkylene- group whereinamido and alkylene are defined herein. Representative amidoalkyl groupsinclude amidomethyl, amidoethyl, dimethylamidomethyl, and the like.

“Amino” as used herein includes a group of formula Y₁Y₂N— wherein Y₁ andY₂ are independently hydrogen, acyl, aryl, or alkyl; or Y₁ and Y₂,together with the nitrogen through which Y₁ and Y₂ are linked, join toform a 4- to 7-membered azaheterocyclyl group (e.g., piperidinyl).Optionally, when Y₁ and Y₂ are independently hydrogen or alkyl, anadditional substituent can be added to the nitrogen, making a quaternaryammonium ion. Representative amino groups include primary amino (H₂N—),methylamino, dimethylamino, diethylamino, tritylamino, and the like.Preferably, “amino” is an —NRR′ group where R and R′ are membersindependently selected from the group consisting of H and alkyl.Preferably, at least one of R and R′ is H.

“Aminoalkyl” as used herein includes an amino-alkylene- group whereinamino and alkylene are defined herein. Representative aminoalkyl groupsinclude aminomethyl, aminoethyl, dimethylaminomethyl, and the like.

“Aroyl” as used herein includes an aryl-CO— group wherein aryl isdefined herein. Representative aroyl include benzoyl, naphth-1-oyl andnaphth-2-oyl.

“Aryl” as used herein includes an aromatic monocyclic or multicyclicring system of 6 to about 14 carbon atoms, preferably of 6 to about 10carbon atoms. Representative aryl groups include phenyl and naphthyl.

“Arylsulfonate ester” as used herein includes an aryl-SO₃— group whereinthe aryl group is as defined herein. Representative arylsulfonate estergroups include phenylsulfonate ester.

An “optionally substituted” arylsulfonate ester includes anarylsulfonate ester as defined herein, wherein the aryl group isadditionally substituted with from 0 to 3 halo, alkyl, aryl, haloalkyl,or haloaryl groups as defined herein. Preferred optionally substitutedarylsulfonate esters include tosylate ester (i.e., p-tolylsulfonateester).

“Aromatic ring” as used herein includes 5-12 membered aromaticmonocyclic or fused polycyclic moieties that may include from zero tofour heteroatoms selected from the group consisting of oxygen, sulfur,selenium, and nitrogen. Exemplary aromatic rings include benzene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, naphthalene,benzathiazoline, benzothiophene, benzofurans, indole, benzindole,quinoline, and the like. The aromatic ring group can be substituted atone or more positions with halo, alkyl, alkoxy, alkoxy carbonyl,haloalkyl, cyano, sulfonato, amino sulfonyl, aryl, sulfonyl,aminocarbonyl, carboxy, acylamino, alkyl sulfonyl, amino and substitutedor unsubstituted substituents.

“Balanced charge” as used herein includes the condition that the netcharge for a compound and its associated counterions be zero understandard physiological conditions. In order to achieve a balancedcharge, a skilled person will understand that after the first additionalsulfonato group that balances the +1 charge of the indolinium ring ofthe compounds herein, a cationic counterion (e.g., the cation of a GroupI metal such as sodium) must be added to balance the negative chargefrom additional sulfonato groups. Similarly, anionic counterions must beadded to balance any additional cationic groups (e.g., most amino groupsunder physiological conditions).

“Biomolecule” as used herein includes a natural or synthetic moleculefor use in biological systems. Preferred biomolecules include a protein,a peptide, an enzyme substrate, a hormone, an antibody, an antigen, ahapten, an avidin, a streptavidin, a carbohydrate, a carbohydratederivative, an oligosaccharide, a polysaccharide, a nucleic acid, adeoxynucleic acid, a fragment of DNA, a fragment of RNA, nucleotidetriphosphates, acyclo terminator triphosphates, PNA, and the like. Morepreferred biomolecules include a protein, a peptide, an antibody, anavidin, a streptavidin, and the like. Even more preferred biomoleculesinclude a peptide, an antibody, an avidin, and a streptavidin.

“Carboxy” and “carboxyl” as used herein include a HOC(O)— group (i.e., acarboxylic acid) or a salt thereof.

“Carboxyalkyl” as used herein includes a HOC(O)-alkylene- group whereinalkylene is defined herein. Representative carboxyalkyls includecarboxymethyl (i.e., HOC(O)CH₂—) and carboxyethyl (i.e., HOC(O)CH₂CH₂—).

“Cycloalkenyl” as used herein includes a cyclic hydrocarbon group of 4to about 15 carbon atoms that contains at least one carbon-carbon doublebond. The cycloalkenyl ring may include from 0 to 6 R¹⁴ substituents and0 to 2 R^(L) substituents, and when present, the ring-fused aryl orheteroaryl rings may also include from 0 to 4 R¹⁴ substituents and 0 to2 R^(L) substituents. Preferred alkenyl groups have 5 to about 12 carbonatoms. More preferred alkenyl groups contain 7 to about 14 carbon atoms.Representative cycloalkenyl groups include cyclopentenyl, cyclohexenyl,and the like.

“Cycloalkynyl” as used herein includes a cyclic hydrocarbon group of 5to about 15 carbon atoms that contains at least one carbon-carbon triplebond. In a preferred aspect, the cyclic hydrocarbon may optionally beinterrupted by a heteroatom (e.g., N, O, S; preferably N) and mayinclude at least one ring-fused aryl or heteroaryl ring (e.g., DBCO orDBCO-1). The cycloalkynyl ring may include from 0 to 6 R¹⁴ substituentsand 0 to 2 R^(L) substituents, and when present, the ring-fused aryl orheteroaryl rings may also include from 0 to 4 R¹⁴ substituents and 0 to2 R^(L) substituents. In some aspect, the R^(L) substituent includes aring-fused heteroaryl group as part of the linking group with thebiomolecule (e.g., the reaction of DBCO with an azide-substitutedbiomolecule). Preferred alkynyl groups have 5 to about 12 carbon atoms.More preferred alkynyl groups contain 7 to about 14 carbon atoms.Representative cycloalkynyl groups include cyclopentynyl, cyclohexynyl,cyclooctynyl, dibenzocyclooctynyl (or DBCO, which includes a nitrogen inthe “octyne” ring or DBCO-1), BARAC, DIFO, DIBO, TMDIBO, DIFO3 and thelike.

“Cycloalkynylcarbonyl” includes the definition of cycloalkynyl abovewith an exocylic carbonyl, for example, a dibenzocyclooctynylcarbonyl orC(O)DBCO, which includes a nitrogen in the “octyne” ring and anexocyclic carbonyl group, and the like.

“Cycloalkyl” as used herein includes a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, preferably of about 5to about 10 carbon atoms. More preferred cycloalkyl rings contain 5 or 6ring atoms. A cycloalkyl group optionally comprises at least onesp²-hybridized carbon (e.g., a ring incorporating an endocyclic orexocyclic olefin). Representative monocyclic cycloalkyl groups includecyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, and the like.Representative multicyclic cycloalkyl include 1-decalin, norbornyl,adamantyl, and the like.

“Cycloalkylene” as used herein includes a bivalent cycloalkyl havingabout 4 to about 8 carbon atoms. Preferred cycloalkylenyl groups include1,2-, 1,3-, or 1,4- cis- or trans-cyclohexylene.

“Cyanine dye” as used herein includes a compound having two substitutedor unsubstituted nitrogen-containing heterocyclic rings joined by anunsaturated bridge. Examples include the structures of Formula I.

“Exocyclic alkene” or “exocyclic olefin” as used interchangeably hereininclude an alkene having one alkene carbon that is part of a ring andthe other alkene carbon not part of the same ring, though it may beincluded within a second ring. The second alkene carbon can beunsubstituted or substituted. If the second alkene carbon isdisubstituted, the substituents can be the same (e.g., 1,1-dimethylsubstitution) or different (e.g., 1-methyl-1-(2-ethoxyethyl)substitution). Examples of compounds with exocyclic alkenes includemethylenecyclohexane; (E)-1-ethylidene-2,3-dihydro-1H-indene;pentan-3-ylidenecycloheptane; 2-cyclobutylidenepropan-1-ol; and(3-methoxycyclopent-2-enylidene)cyclohexane.

“Geminal” substituents as used herein includes two or more substituentsthat are directly attached to the same atom. An example is 3,3-dimethylsubstitution on a cyclohexyl or spirocyclohexyl ring.

“Halo” or “halogen” as used herein include fluoro, chloro, bromo, oriodo.

“Haloalkyl” as used herein includes an alkyl group wherein the alkylgroup includes one or more halo- substituents.

“Haloaryl” as used herein includes an alkyl group wherein the aryl groupincludes one or more halo- substituents.

“Heptamethine” as used herein includes a polymethine containing sevenpolymethine carbons. In a preferred embodiment, the heptamethine issubstituted at the 4-position.

“Heteroatom” as used herein includes an atom other than carbon orhydrogen. Representative heteroatoms include O, S, and N. The nitrogenor sulfur atom of the heteroatom is optionally oxidized to thecorresponding N-oxide, S-oxide (sulfoxide), or S,S-dioxide (sulfone). Ina preferred aspect, a heteroatom has at least two bonds to alkylenecarbon atoms (e.g., —C₁-C₉ alkylene-O—C₁-C₉ alkylene-). In someembodiments, a heteroatom is further substituted with an acyl, alkyl,aryl, cycloalkyl, heterocyclyl, or heteroaryl group (e.g., —N(Me)—;—N(Ac)-).

“Heteroaroyl” as used herein includes a heteroaryl-C(O)— group whereinheteroaryl is as defined herein. Representative heteroaroyl groupsinclude thiophenoyl, nicotinoyl, pyrrol-2-ylcarbonyl, pyridinoyl, andthe like.

“Heterocycloyl” as used herein includes a heterocyclyl-C(O)— groupwherein heterocyclyl is as defined herein. Representative heterocycloylgroups include N-methyl prolinoyl, tetrahydrofuranoyl, and the like.

“Heterocyclyl” as used herein includes a non-aromatic saturatedmonocyclic or multicyclic ring system of about 3 to about 10 ring atoms,preferably about 5 to about 10 ring atoms, in which one or more of theatoms in the ring system is an element or elements other than carbon,e.g., nitrogen, oxygen or sulfur. Preferred heterocyclyl groups containabout 5 to about 6 ring atoms. A heterocyclyl group optionally comprisesat least one sp²-hybridized atom (e.g., a ring incorporating ancarbonyl, endocyclic olefin, or exocyclic olefin). The prefix “aza,”“oxa,” or “thia” before heterocyclyl means that at least a nitrogen,oxygen or sulfur atom respectively is present as a ring atom. Thenitrogen or sulfur atom of the heterocyclyl is optionally oxidized tothe corresponding N-oxide, S-oxide or S,S-dioxide. Representativemonocyclic heterocyclyl rings include piperidyl, pyrrolidinyl,piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl,1,3-dioxolanyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl,tetrahydrothiopyranyl, and the like.

“Heterocyclylene” as used herein includes a bivalent heterocyclyl group.Representative cycloalkylenyl groups include 1,2-, 1,3-, or 1,4-piperidinylene as well as 2,3- or 2,4- cis- or trans-piperidinylene.

“Heteroaryl” as used herein includes an aromatic monocyclic ormulticyclic ring system of about 5 to about 14 ring atoms, preferablyabout 5 to about 10 ring atoms, in which at least one of the atoms inthe ring system is an element other than carbon, i.e., nitrogen, oxygenor sulfur. Preferred heteroaryls contain about 5 to about 6 ring atoms.The prefix “aza,” “oxa,” or “thia” before heteroaryl means that at leasta nitrogen, oxygen or sulfur atom respectively is present as a ringatom. A nitrogen atom of a heteroaryl is optionally oxidized to thecorresponding N-oxide. Representative heteroaryls include pyrazinyl,furanyl, thienyl, pyridyl, pyrimidinyl, isoxazolyl, isothiazolyl,oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl,triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl,phthalazinyl, imidazo[1,2-a]pyridine, imidazo[2,1-b]thiazolyl,benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl,quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl,pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl,1,2,4-triazinyl, benzothiazolyl and the like.

“Hydroxyalkyl” as used herein includes an alkyl group as defined hereinsubstituted with one or more hydroxy groups. Preferred hydroxyalkylscontain lower alkyl. Representative hydroxyalkyl groups includehydroxymethyl and 2-hydroxyethyl.

When any two substituent groups or any two instances of the samesubstituent group are “independently selected” from a list ofalternatives, they may be the same or different. For example, if R^(a)and R^(b) are independently selected from the group consisting ofmethyl, hydroxymethyl, ethyl, hydroxyethyl, and propyl, then a moleculewith two R^(a) groups and two R^(b) groups could have all groups bemethyl. Alternatively, the first R^(a) could be methyl, the second R^(a)could be ethyl, the first R^(b) could be propyl, and the second R^(b)could be hydroxymethyl (or any other substituents taken from the group).Alternatively, both R^(a) and the first R^(b) could be ethyl, while thesecond R^(b) could be hydroxymethyl (i.e., some pairs of substituentgroups may be the same, while other pairs may be different).

“Linking group” as used herein includes the atoms joining a compound ofFormula I with a biomolecule. Table 1 includes a list of preferred bondsfor linking groups (i.e., Column C); the linking group comprises theresulting bond and optionally can include additional atoms. See also R.Haugland, Molecular Probes Handbook of Fluorescent Probes and ResearchChemicals, Molecular Probes, Inc. (1992). In one embodiment, R¹⁶represents a linking group precursor before the attachment reaction witha biomolecule, and R^(L) represents the resultant attachment between thecompound of Formula I and the biomolecule (i.e., R^(L) comprises thelinking group and the biomolecule linked thereby). Preferred reactivefuntionalities include phosphoramidite groups, an activated ester (e.g.,an NHS ester), thiocyanate, isothiocyanate, maleimide and iodoacetamide.

“Methine carbon” or “polymethine carbon” as used herein include a carbonthat is directly connecting the two heterocyclic rings by means of thepolymethine bridge. In a preferred embodiment, at least one polymethinecarbon of a polymethine bridge is additionally substituted with anothergroup such as alkyl, cycloalkyl, or aryl (e.g., —CH═CH—C(Ar)═CH—CH═ or═CH—CH═C(Ar)-(CH═CH)₂—).

“Oxo” as used herein includes a group of formula >C═O (i.e., a carbonylgroup —C(O)—).

“Pentamethine” as used herein includes a polymethine containing fivepolymethine carbons. In a preferred embodiment, the pentamethine issubstituted at the 3-position.

A “photoactivatable moiety” is a chemical group or molecule that, uponexposure to light, absorbs a photon to enter an excited state. Theexcited-state group or molecule undergoes a chemical reaction or seriesof reactions. Alternatively, the excitation changes the light-emittingproperties of the group or molecules (e.g., photoactivatable fluorescentdyes). Examples of photoactivatable moieties include aryl azides,benzophenones (e.g., 4-benzoyloxybenzoic acid as well as its esters andamides), nitroaryl groups (e.g., 5-carboxymethoxy-2-nitrobenzyl (CMNB);α-carboxy-2-nitrobenzyl (CNB); 4,5-dimethoxy-2-nitrobenzyl (DMNB);1-(4,5-dimethoxy-2-nitrophenyl)ethyl (DMNPE); nitrophenyl (NP); and1-(2-nitrophenyl)ethyl (NPE) groups), coumarins, diazo groups,photoactivatable fluorescent dyes (e.g.,5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether,β-alanine-carboxamide, succinimidyl ester), and tetrazoles.

“Polyene” as used herein includes a straight or branched bivalenthydrocarbon chain containing at least two “alkenylene” groups as definedherein that are in conjugation. The polyene is optionally substitutedwith one or more “alkylene group substituents” as defined herein. Aportion of the polyene may be incorporated into a ring (i.e., ═C(R)—,wherein R and the terminal bond are linked in a larger ring; or—C(R¹)═C(R²)—, wherein R¹ and R² are linked in a larger ring).Representative polyenes include —CH═CH—CH═CH—, —CH═CH—C(Ar)═CH—CH═C(R)—,—C(R)═CH—CH═C(Ar)-(CH═CH)₂—, and the like.

“Polymethine” or “polymethine bridge” as used herein includes the seriesof conjugated, sp²-hybridized carbons that form the unsaturated bridgedirectly connecting the two nitrogen-containing heterocyclic rings of acompound of Formula I. In a preferred embodiment, the polymethine hasfive or seven carbons directly connecting the heterocyclic rings (i.e.,pentamethine or heptamethine).

“Phosphoramidityl” as used herein includes a trivalent phosphorous atombonded to two alkoxy groups and an amino group.

“Spirocycloalkyl” as used herein includes a cycloalkyl in which geminalsubstituents on a carbon atom are replaced to form a 1,1-substitutedring; a spirocycloalkynyl is a cycloalkynyl in which geminalsubstituents on a carbon atom are replaced to form a 1,1-substitutedring. A preferred example is BCN.

“Sulfonato” as used herein includes an —SO₃ ⁻ group, preferably balancedby a cation such as H⁺, Na⁺, K⁺, and the like.

“Sulfonatoalkyl” as used herein includes an sulfonato-alkylene- groupwherein sulfonato and alkylene are as defined herein. A more preferredembodiment includes alkylene groups having from 2 to 6 carbon atoms, anda most preferred embodiment includes alkylene groups having 2, 3, or 4carbons. Representative sulfonatoalkyls include sulfonatomethyl,3-sulfonatopropyl, 4-sulfonatobutyl, 5-sulfonatopentyl,6-sulfonatohexyl, and the like.

In general, the unit prefix “u” as used herein is equivalent to “μ” or“micro.” For example, “ul” is equivalent to “μl” or “microliters.”

Cyanine Dye Compounds

In one embodiment, the present invention provides a compound of FormulaI:

wherein Q is a member selected from the group of one-methine-carbonsegment and three-methine-carbon segments:

respectively; wherein the segment is the central portion of either afive- or a seven-methine-carbon polymethine bridge.

In a preferred aspect, Q is a portion of a polymethine bridge that is apentamethine:

More preferably, Q is

In a second preferred aspect, Q is a portion of a polymethine bridgethat is a heptamethine:

More preferably, Q is

In an alternative preferred aspect, Q is a portion of a polymethinebridge that is a substituted heptamethine:

More preferably, Q is

In an alternative, more preferred aspect, the substituted heptamethineincludes a cycloalkyl ring:

Still more preferably, Q is

In a third preferred aspect, Q is selected from the group consisting of:

More preferably, Q is

In a fourth preferred aspect, Q is selected from the group consistingof:

Each R¹ is a member selected from the group consisting of L-Y—Z and analkyl group that is additionally substituted with from 0 to 1 R¹³ andfrom 0 to 1 R¹⁶; wherein the alkyl is optionally interrupted by at leastone heteroatom.

In a preferred aspect, R¹ is C₁-C₂₀ alkyl. In a more preferred aspect,R¹ is C₁-C₁₂ or C₂-C₈ alkyl. In a still more preferred aspect, R¹ isC₂-C₆ alkyl. In a yet still more preferred aspect, R¹ is ethyl, propyl,butyl, or pentyl, and R¹ is additionally substituted with 1 R¹³.

In a preferred aspect, R¹ is not interrupted by a heteroatom.Alternatively, R¹ is interrupted by at least one ether, thioether,substituted amino, or amido group.

In another preferred aspect, R¹ is (CH₂)_(r)SO₃H or (CH₂)_(r)SO₃ ⁻; andr is an integer from 1 to 20. In a more preferred aspect, r is 2, 3, or4.

In still another preferred aspect, R¹ is an alkyl group that isadditionally substituted with 1 R¹³ that is selected from the group ofhydroxyl, amino, carboxy, and sulfonato. In a more preferred aspect, theR¹³ substituent of R¹ is carboxy or sulfonato. In a still more preferredaspect, the R¹³ substituent of R¹ is sulfonato. In a yet still morepreferred aspect, R¹ is sulfonatoethyl, sulfonatopropyl, sulfonatobutyl,or sulfonatopentyl.

In yet another preferred aspect, R¹ is an unbranched alkyl group that isadditionally substituted with 1 R¹³. In a more preferred aspect, R¹ isan unbranched alkyl group that is substituted with R¹³ at the end of thealkyl group opposite to its attachment point to the cyanine dyeheterocyclic nitrogen. In a still more preferred aspect, R¹ is2-sulfonatoethyl, 3-sulfonatopropyl, 4-sulfonatobutyl, or5-sulfonatopentyl. In a yet still more preferred aspect, R¹ is3-sulfonatopropyl or 4-sulfonatobutyl; more preferably, R¹ is3-sulfonatopropyl.

In still another preferred aspect, R¹ is L-Y—Z. For example, L is aC₁-C₂₀ alkylene group such as C₂-C₈ alkylene; Y is a C(O)NH group; and Zis L-R¹⁶, wherein L is a C₁-C₂₀ alkylene group such as C₂-C₈ alkyleneand R¹⁶ is a cycloalkynylcarbonyl like C(O)DBCO (see for example,compound 66). In another aspect, R¹ is L-Y—Z, wherein L is a C₁-C₂₀alkylene group such as C₂-C₈ alkylene; Y is a C(O)NH group; and Z isL-R¹⁶, wherein L is a C₁-C₂₀ alkylene group such as C₂-C₈ alkyleneoptionally interrupted by a heteroatom (e.g., ((CH₂CH₂O)₃—CH₂CH₂—) andR¹⁶ is an azido group (see for example, compound 67).

Each R^(2a) and R^(2b) is a member independently selected from the groupconsisting of alkyl, alkenyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl,amidoalkyl, alkylthioalkyl, carboxyalkyl, alkoxycarbonylalkyl, orsulfonatoalkyl; wherein a carbon of the member is additionallysubstituted with from 0 to 1 R¹⁶; or, alternatively, a R^(2a) and R^(2b)pair, together with the ring carbon to which the R^(2a) and R^(2b) arebonded, join to form a spirocycloalkyl ring, wherein the spirocycloalkylring is additionally substituted with from 0 to 6 R¹⁴ and from 0 to 1R¹⁶, or an exocyclic alkene, wherein the exocyclic alkene isadditionally substituted with from 0 to 2 R¹⁴ and from 0 to 1 R¹⁶.

In a preferred aspect, all R^(2a) are the same substituent.Alternatively, all R^(2b) are the same substituent. More preferably, allR^(2a) are the same substituent, and all R^(2b) are the samesubstituent.

In another preferred aspect, R^(2a) and R^(2b) are the same. In a morepreferred aspect, R^(2a) and R^(2b) are alkyl, alkenyl, aminoalkyl,carboxyalkyl, or sulfonatoalkyl. In a still more preferred aspect,R^(2a) and R^(2b) are alkyl, carboxyalkyl, or sulfonatoalkyl. In a yetstill more preferred aspect, R^(2a) and R^(2b) are methyl.

In an alternative aspect, R^(2a) and R^(2b) are different. In a morepreferred aspect, R^(2a) is alkyl, and R^(2b) is selected from the groupof alkyl, alkenyl, aminoalkyl, carboxyalkyl, or sulfonatoalkyl. In astill more preferred aspect, R^(2a) is alkyl, and R^(2b) is selectedfrom the group of alkyl, carboxyalkyl, or sulfonatoalkyl. Yet still morepreferably, R^(2a) is methyl.

In another preferred aspect, R^(2a) and R^(2b), together with the ringcarbon to which R^(2a) and R^(2b) are bonded, join to form aspirocycloalkyl ring, wherein the spirocycloalkyl ring is additionallysubstituted with from 0 to 6 R¹⁴. In a more preferred aspect, R^(2a) andR^(2b) form a cyclopentyl or sulfonatoalkyl. In a still more preferredaspect, R¹⁴ is alkyl. In a yet still more preferred aspect, R¹⁴ ismethyl.

In an alternative aspect, R^(2a) and R^(2b), together with the ringcarbon to which R^(2a) and R^(2b) are bonded, join to form an exocyclicalkene, wherein the exocyclic alkene is additionally substituted withfrom 0 to 2 R¹⁴. In a more preferred aspect, the exocyclic alkene issymmetrically substituted (e.g., unsubstituted; dialkyl; dicyano).Alternatively, the exocyclic alkene is substituted with two R¹⁴ groups.Still more preferably, the exocylic alkene's R¹⁴ substituent is cyano.

In an alternative preferred aspect, R^(2a) and R^(2b), together with theatom to which R^(2a) and R^(2b) are bonded, join to form aspirocycloalkyl ring. In a more preferred aspect, R^(2a) and R^(2b) forma cyclopentyl or cyclohexyl ring. In an alternative more preferredaspect, R^(2a) and R^(2b) form a cyclopentyl or cyclohexyl ringadditionally substituted with from 0 to 6 R¹⁴. In a still more preferredaspect, R¹⁴ is alkyl.

Alternatively and preferably, the spirocycloalkyl ring has at least onepair of geminal R¹⁴ alkyl substituents. More preferably, these geminalR¹⁴ substituents are methyl (e.g., 3,3- or 4,4-dimethyl substitution).

Each R³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and R^(6b) is a memberindependently selected from the group consisting of hydrogen, alkyl,alkenyl, halo, hydroxyl, alkoxy, amino, cyano, carboxyl, alkoxycarbonyl,amido, sulfonato, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, andsulfonatoalkyl; wherein a carbon of the member is additionallysubstituted with from 0 to 1 R¹⁶; or, alternatively, a pair of saidmembers that is selected from the group consisting of R³ and R^(4a), anR^(4a) and R^(5a), and an R^(5a) and R^(6a), together with the pair ofatoms to which the pair of said members is bonded, joins to form an arylring, wherein the aryl ring is additionally substituted with from 0 to 2R¹⁴ and from 0 to 1 R¹⁶.

In a first aspect, each R³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), andR^(6b) is a member independently selected from the group consisting ofhydrogen, alkyl, alkenyl, halo, alkoxy, cyano, carboxyl, alkoxycarbonyl,amido, sulfonato, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, andsulfonatoalkyl. In a preferred aspect, each R³, R^(4a), R^(4b), R^(5a),R^(5b), R^(6a), and R^(6b) is a member independently selected from thegroup of hydrogen, alkyl, carboxy, carboxyalkyl, halo, sulfanato, andsulfanatoalkyl. In a more preferred embodiment, each R³, R^(4a), R^(4b),R^(5a), R^(5b), R^(6a), and R^(6b) is a member independently selectedfrom the group of hydrogen, halo, and sulfanato.

In an alternative aspect, at least one member of the group R³, R^(4a),R^(4b), R^(5a), R^(5b), R^(6a), and R^(6b) is hydrogen. Alternatively,exactly one member of the group R³, R^(4a), R^(4b), R^(5a), R^(5b),R^(6a), and R^(6b) is hydrogen. In a preferred aspect, at least one pairof substituents selected from the pairs R³ and R^(4a); R³ and R^(5a); R³and R^(6a); R^(4a) and R^(5a); R^(4a) and R^(6a); R^(5a) and R^(6a), R³and R^(4b); R³ and R^(5b); R³ and R^(6b); R^(4b) and R^(5b); R^(4b) andR^(6b); and R^(5b) and R^(6b) is hydrogen. Alternatively, exactly two,exactly three, exactly four, exactly five, or exactly six members of thegroup R³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and R^(6b) arehydrogen. In a more preferred aspect, exactly four members of the groupR³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and R^(6b) are hydrogen.Alternatively, exactly five members of the group R³, R^(4a), R^(4b),R^(5a), R^(5b), R^(6a), and R^(6b) are hydrogen. In a still morepreferred aspect, R³, R^(4a), R^(6a), R^(4b), R^(6b) are hydrogen.

In another alternative aspect, at least one member of the group R³,R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and R^(6b) is sulfonato orsulfonatoalkyl. Alternatively, exactly one substituent selected from thegroup R³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and R^(6b) issulfonato or sulfonatoalkyl. In a preferred aspect, R^(5a) is sulfonato.In still another aspect, both members of a pair of substituents selectedfrom the pairs R³ and R^(4a); R³ and R^(5a); R³ and R^(6a); R^(4a) andR^(5a); R^(4a) and R^(6a); R^(5a) and R^(6a), R³ and R^(4b); R³ andR^(5b); R³ and R^(6b); R^(4b) and R^(5b); R^(4b) and R^(6b); and R^(5b)and R^(6b) are each a member independently selected from the group ofsulfonato or sulfonatoalkyl. Alternatively, exactly two, exactly three,exactly four, exactly five, or exactly six members of the group R³,R^(4a); R^(4b), R^(5a), R^(5b), R^(6a), and R^(6b) are each a memberindependently selected from the group of sulfonato or sulfonatoalkyl.

In another alternative aspect, at least one member of the group R³,R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and R^(6b) is anionic atphysiological pH (e.g., sulfonato —SO₃ ⁻, carboxyl —CO₂ ⁻).Alternatively, exactly one member of the group R³, R^(4a), R^(4b),R^(5a), R^(5b), R^(6a), and, and R^(6b) is anionic at physiological pH.In a preferred aspect, R^(5a) is anionic at physiological pH. In stillanother aspect, each member of a pair of substituents selected from thepairs R³ and R^(4a); R³ and R^(5a); R³ and R^(6a); R^(4a) and R^(5a);R^(4a) and R^(6a); R^(5a) and R^(6a), R³ and R^(4b); R³ and R^(5b); R³and R^(6b); R^(4b) and R^(5b); R^(4b) and R^(6b); and R^(5b) and R^(6b)is anionic at physiological pH. Alternatively, exactly two, exactlythree, exactly four, exactly five, or exactly six members of the groupR³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and, and R^(6b) are anionicat physiological pH. exactly two, exactly three, or exactly four membersof the group R³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and, and R^(6b)are anionic at physiological pH.

In another alternative aspect, at least one member of the group R³,R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and, and R^(6b) is halo.Alternatively, exactly one substituent selected from the group R³,R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and, and R^(6b) is halo. In apreferred aspect, R^(5b) is halo; more preferably, R^(5b) is chloro. Instill another aspect, both members of a pair of substituents selectedfrom the pairs R³ and R^(4a); R³ and R^(5a); R³ and R^(6a); R^(4a) andR^(5a); R^(4a) and R^(6a); R^(5a) and R^(6a), R³ and R^(4b); R³ andR^(5b); R³ and R^(6b); R^(4b) and R^(5b); R^(4b) and R^(6b); and R^(5b)and R^(6b) are each an independently selected halo. Alternatively,exactly two, exactly three, exactly four, exactly five, or exactly sixmembers of the group R³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and,and R^(6b) are each an independently selected halo.

In a second aspect, a pair of members that is selected from the group ofR³ and R^(4a), R^(4a) and R^(5a), R^(5a) and R^(6a), R^(4b) and R^(5b),and R^(5b) and R^(6b), together with the pair of atoms to which the pairof members is bonded, joins to form an aryl ring (i.e., the aryl ringformed from R^(n) and R^(n+1)), wherein the aryl ring is additionallysubstituted with from 0 to 2 R¹⁴. In a preferred aspect, the pair ofmembers R^(5a) and R^(6a) or R^(5b) and R^(6b), together with the pairof atoms to which the pair of members is bonded, joins to form a phenylring that is additionally substituted with from 0 to 2 R¹⁴. In a morepreferred aspect, the phenyl ring is additionally substituted with from1 to 2 R¹⁴. In a still more preferred aspect, the phenyl ring isadditionally substituted with 2 R¹⁴.

In a preferred aspect, the R¹⁴ substituents of the aryl ring formed fromR^(n) and R^(n+1) (e.g., the aryl ring formed from R^(5a) and R^(6a))are carboxy, carboxyalkyl, halo, sulfonato, or sulfonatoalkyl. In astill more preferred aspect, the R¹⁴ substituents are sulfonato orsulfonatoalkyl. In a yet still more preferred aspect, the benzindoliniumR¹⁴ substituents are sulfonato.

In a more preferred aspect, the aryl ring formed from R^(n) and R^(n+1)is additionally substituted with from 1 to 2 R¹⁴, and a R¹⁴ substituentof the aryl ring is attached to a carbon adjacent to the ring junctionwith the indolinium ring. Alternatively, the aryl ring is additionallysubstituted with from 1 to 2 R¹⁴, and a R¹⁴ substituent of the aryl ringis attached to a carbon non-adjacent to the ring junction with theindolinium ring. Alternatively, the aryl ring is additionallysubstituted with one adjacent substituent and one non-adjacentsubstituent (e.g., the compound of Formula Ia).

Alternatively, in a preferred aspect, the compound has Formula Ia:

In a still more preferred aspect, the benzindolinium R¹⁴ substituents ofFormula Ia are carboxy, carboxyalkyl, halo, sulfonato, orsulfonatoalkyl. In a still more preferred aspect, the benzindolinium R¹⁴substituents are sulfonato or sulfonatoalkyl. In a yet still morepreferred aspect, the benzindolinium R¹⁴ substituents are sulfonato.

Each R⁷ is a member independently selected from the group consisting ofhydrogen and alkyl; wherein the alkyl is additionally substituted withfrom 0 to 3 R¹⁴ and from 0 to 1 R¹⁶; or, alternatively, both R⁷,together with the intervening segment of the polyene to which both R⁷are bonded, join to form a ring, wherein said ring is selected from thegroup consisting of a cycloalkyl and a heterocyclyl ring; wherein thering is additionally substituted with from 0 to 3 R¹⁴ and from 0 to 1R¹⁶.

In one aspect, both R⁷, together with the intervening segment of thepolyene to which both R⁷ are bonded, join to form a ring selected fromthe group of a five-membered ring and a six-membered ring, wherein thering is additionally substituted with from 0 to 3 R¹⁴. In a morepreferred aspect, the ring is a six-membered ring. In a still morepreferred aspect, the ring is cyclohexyl (i.e., both R⁷ combine to forma —(CH₂)₃— linking group). In an alternative more preferred aspect, thering is a five-membered ring. In a still more preferred aspect, the ringis cyclopentyl (i.e., both R⁷ combine to form a —(CH₂)₂— linking group).

R⁸, R⁹, R¹⁰, R¹¹ and R¹² are each a member independently selected fromthe group consisting of hydrogen, alkyl, alkenyl, halo, alkoxy,sulfonato, sulfonatoalkyl, hydroxyl, amino, carboxyl, alkoxycarbonyl,cyano, amido, thioacetyl, and -L-Y—Z; wherein, if present, at least onemember selected from the group consisting of R⁸, R⁹, and R¹⁰ is -L-Y—Z.

In one aspect, R⁸ is -L-Y—Z. Preferably, R⁹, R¹⁰, R¹¹, and R¹² are eacha member independently selected from the group consisting of hydrogen,alkyl, halo, sulfonato, and sulfonatoalkyl.

In a second aspect, R⁸ is hydrogen, alkyl, alkoxy, or halo. In a morepreferred aspect, R⁸ is hydrogen.

Alternatively, R⁸ is a carboxyalkyl. Preferably, R⁸ is a lower alkylgroup with a carboxyl substituent. More preferably, R⁸ is5-carboxypentyl, 4-carboxybutyl, 3-carboxypropyl, 2-carboxyethyl, orcarboxymethyl. Still more preferably, R⁸ is 5-carboxypentyl or2-carboxyethyl.

Alternatively, R⁸ is carboxyl, alkoxycarbonyl, or amido; morepreferably, R⁸ is carboxyl.

In one aspect, R¹⁰ is -L-Y—Z. Preferably, R⁸, R⁹, R¹¹, and R¹² are eacha member independently selected from the group consisting of hydrogen,alkyl, halo, sulfonato, and sulfonatoalkyl.

In a second aspect, R¹⁰ is hydrogen, alkyl, alkoxy, or halo. In a morepreferred aspect, R¹⁰ is hydrogen.

Alternatively, R¹⁰ is a carboxyalkyl. Preferably, R¹⁰ is a lower alkylgroup with a carboxyl substituent. More preferably, R¹⁰ is5-carboxypentyl, 4-carboxybutyl, 3-carboxypropyl, 2-carboxyethyl, orcarboxymethyl. Still more preferably, R¹⁰ is 5-carboxypentyl or2-carboxyethyl.

Alternatively, R¹⁰ is carboxyl, alkoxycarbonyl, or amido; morepreferably, R¹⁰ is carboxyl.

In one aspect, R⁹ is -L-Y—Z. Preferably, R⁸, R¹⁰, R¹¹, and R¹² are eacha member independently selected from the group consisting of hydrogen,alkyl, halo, sulfonato, and sulfonatoalkyl.

In a second aspect, R⁹ is hydrogen, alkyl, alkoxy, or halo. In a morepreferred aspect, R⁹ is hydrogen.

Alternatively, R⁹ is a carboxyalkyl. Preferably, R⁹ is a lower alkylgroup with a carboxyl substituent. More preferably, R⁹ is5-carboxypentyl, 4-carboxybutyl, 3-carboxypropyl, 2-carboxyethyl, orcarboxymethyl. Still more preferably, R⁹ is 5-carboxypentyl or2-carboxyethyl.

Alternatively, R⁹ is carboxyl, alkoxycarbonyl, or amido; morepreferably, R⁹ is carboxyl.

In one aspect, R¹¹ and R¹² are each a member independently selected fromthe group of hydrogen, alkyl, alkenyl, halo, alkoxy, sulfonato,hydroxyl, amino, carboxyl, alkoxycarbonyl, cyano, amido, thioacetyl, and-L-Y—Z. Preferably, R¹¹ and R¹² are each a member independently selectedfrom the group of hydrogen, alkyl, halo, sulfonato, and sulfonatoalkyl.More preferably, R¹¹ and R¹² are each a member independently selectedfrom the group of hydrogen, halo, and sulfonato.

In a second aspect, R¹¹ is hydrogen, alkyl, alkoxy, or halo. In a morepreferred aspect, R¹¹ is hydrogen. In a still more preferred aspect, R¹⁰and R¹¹ are hydrogen.

In a third aspect, R¹² is hydrogen, alkyl, alkoxy, or halo. In a morepreferred aspect, R¹² is hydrogen. In a still more preferred aspect, R¹⁰and R¹² are hydrogen. Alternatively, R¹¹ and R¹² are hydrogen. In a yetstill more preferred aspect, R¹⁰, R¹¹, and R¹² are hydrogen.

In a fourth aspect, the phenyl ring substituted with R⁸, R⁹, R¹⁰, R¹¹,and R¹² is 1,2,3-substituted with independently selected substituentsother than hydrogen, and the 1-substituent is the polymethine bridge(e.g., R⁸ is -L-Y—Z and R⁹ is alkyl; R⁸ is halo- and R⁹ is -L-Y—Z).Alternatively, the ring is 1,2,4-substituted. Alternatively, the ring is1,2,5-substituted. Alternatively, the ring is 1,2,6-substituted.Alternatively, the ring is 1,3,4-substituted. Alternatively, the ring is1,3,5-substituted. Alternatively, the ring is 1,3,6-substituted.

In a fifth aspect, the phenyl ring substituted with R⁸, R⁹, R¹⁰, R¹¹,and R¹² is 1,2,3,4-substituted with independently selected substituentsother than hydrogen, and the 1-substituent is the polymethine bridge.Alternatively, the ring is 1,2,3,5-substituted. Alternatively, the ringis 1,2,3,6-substituted. Alternatively, the ring is 1,2,4,5-substituted.Alternatively, the ring is 1,2,4,6-substituted. Alternatively, the ringis 1,2,5,6-substituted. Alternatively, the ring is 1,3,4,5-substituted.Alternatively, the ring is 1,3,4,6-substituted. Alternatively, the ringis 1,3,5,6-substituted.

In a sixth aspect, the phenyl ring substituted with R⁸, R⁹, R¹⁰, R¹¹,and R¹² is 1,2,3,4,5-substituted with independently selectedsubstituents other than hydrogen, and the 1-substituent is thepolymethine bridge. Alternatively, the ring is 1,3,4,5,6-substituted.Alternatively, the ring is 1,2,4,5,6-substituted. Alternatively, thering is 1,2,3,5,6-substituted. Alternatively, the ring is1,2,3,4,6-substituted. Alternatively, the ring is independentlysubstituted at each ring position.

In a seventh aspect, R⁸, R¹⁰, R¹¹, and R¹² are each a memberindependently selected from the group of hydrogen, alkyl, halo,sulfonato, and sulfonatoalkyl.

In an eighth aspect, the combination of the phenyl ring and itssubstituents R⁸, R⁹, R¹⁰, R¹¹, and R¹² has at least ten carbons.

Each R¹³ is a member independently selected from the group of hydroxyl,amino, carboxyl, alkoxycarbonyl, amido, sulfonato, and thioacetyl. In apreferred embodiment, R¹³ is carboxyl, amido, or alkoxycarbonyl. In amore preferred embodiment, R¹³ is carboxyl. Alternatively, R¹³ issulfonato.

Each R¹⁴ is a member independently selected from the group consisting ofalkyl, alkenyl, halo, hydroxyl, alkoxy, amino, cyano, carboxyl,alkoxycarbonyl, amido, sulfonato, alkoxycarbonylalkyl, and alkoxyalkyl;wherein the alkyl or alkenyl is additionally substituted with from 0 to1 R¹³ and from 0 to 1 R¹⁶. In a preferred aspect, R¹⁴ is alkyl, alkenyl,carboxyl, alkoxycarbonyl, amido, alkoxycarbonylalkyl, or halo.Alternatively, R¹⁴ is carboxyalkyl, hydroxyalkyl, halo, orsulfonatoalkyl. In a more preferred aspect, R¹⁴ is alkyl or alkylsubstituted with 1 R¹³. Alternatively, R¹⁴ is halo or sulfonato.

Each L is an optional member independently selected from the groupconsisting of a bond, a C₁-C₂₀ alkylene, and a C₁-C₂₀ alkenylene;wherein the alkylene or alkenylene is optionally interrupted by at leastone heteroatom. In a preferred aspect, L is a bond. Alternatively, L isa C₁-C₁₄ alkylene; more preferably, L is a C₁-C₁₀ alkylene or a C₁-C₆alkylene. Alternatively, L is a C₁-C₁₂ alkylene interrupted by etherlinkages (e.g., a polyethylene glycol oligomer).

In a preferred aspect, the alkylene or alkenylene is not interrupted bya heteroatom. Alternatively, L is interrupted by at least one ether,thioether, substituted amino, or amido group.

Each Y is an optional member independently selected from the groupconsisting of a bond, —O—, —S—, —NH—, —NHC(O)—, —C(O)NH—, —NR¹⁵—,—NR¹⁵C(O)—, —C(O)NR¹⁵—, —N(Z)—, —N(Z)C(O)—, and —C(O)N(Z)—. In apreferred aspect, Y is a bond. Alternatively, Y is —O—. Alternatively, Yis an amido group optionally substituted with R¹⁵ at the amido nitrogen.

Each Z is independently selected from the group consisting of -L-R¹³ and-L-R¹⁶. In a preferred aspect, the -L- is a C₁-C₂₀ alkylene; morepreferably, a C₁-C₁₂ alkylene; and still more preferably, a C₁-C₁₀alkylene. Alternatively, the -L- is a bond. Yet still more preferably,the -L- is C₁-C₆ alkyl. Alternatively, the -L- is interrupted by etherlinkages (e.g., a polyethylene glycol oligomer). In a still morepreferred aspect, Z is carboxyalkyl or sulfonatoalkyl. In a yet stillmore preferred aspect, Z is 5-carboxypentyl or 4-carboxybutyl.

In an alternative preferred aspect, Z is a carboxyalkyl. Preferably, Zis a lower alkyl group with a carboxy- substituent. More preferably, Zis 5-carboxypentyl, 4-carboxybutyl, 3-carboxypropyl, 2-carboxyethyl, orcarboxymethyl. Still more preferably, Z is 5-carboxypentyl or2-carboxyethyl.

In another alternative preferred aspect, -L-Y— is (CH₂)_(t); Z iscarboxyl or activated acyl; and t is an integer from 0 to 10.

In still another alternative preferred aspect, -L-Y— is a bond. Morepreferably, Z is directly bonded to the phenyl ring or the polymethylenebridge.

In still another alternative preferred aspect, the Z group's L group isa bond, and R¹³ or R¹⁶ is connected directly to -L-Y— or directly bondedto the phenyl ring itself if L and Y are absent.

In yet still another alternative preferred aspect, -L-Y—Z has at leastthree carbons. Alternatively, Z has at least three carbons.

In an alternative embodiment, —Y—Z is a member selected from the groupconsisting of —N(Z)₂, —N(Z)C(O)Z, and —C(O)N(Z)₂, and the two Z groupsmay optionally be linked to form a cycloalkynyl group. Examples of—N(Z)₂ cycloalkynyl groups are DBCO or DBCO-1 illustrated as follows:

Each R¹⁵ is a member independently selected from the group consisting ofalkyl and alkoxycarbonylalkyl; wherein the alkyl is optionallyinterrupted by at least one heteroatom. In a preferred aspect, R¹⁵ isalkyl. In a more preferred aspect, R¹⁵ is lower alkyl. Alternatively,R¹⁵ is interrupted by ether linkages (e.g., a polyethylene glycololigomer).

In a preferred aspect, the alkyl is not interrupted by a heteroatom. Ina preferred aspect, R¹⁵ is alkyl. In a more preferred aspect, R¹⁵ islower alkyl.

Alternatively, L is interrupted by at least one ether, thioether,substituted amino, or amido group. Preferably, R¹⁵ is interrupted by atleast one ether group (e.g., a polyethylene glycol oligomer).

Each R¹⁶ is independently a member selected from the group consisting ofactivated acyl, acrylamido, optionally substituted alkylsulfonate ester,azido, optionally substituted arylsulfonate ester, optionallysubstituted amino, aziridino, boronato, cycloalkynyl,cycloalkynylcarbonyl, diazo, formyl, glycidyl, halo, haloacetamidyl,haloalkyl, haloplatinato, halotriazino, hydrazinyl, imido ester,isocyanato, isothiocyanato, maleimidyl, mercapto, phosphoramidityl, aphotoactivatable moiety, vinyl sulfonyl, alkynyl, a pegylated azido, apegylated alkynyl, a pegylated cycloalkynyl, an ortho substitutedphosphinyl aryl ester (e.g., TPPME), a spirocycloalkynyl, and an orthosubstituted phosphine oxide aryl ester.

In certain aspects, R¹⁶ has the following structures:

In a preferred aspect, R¹⁶ is activated acyl, maleimidyl,phosphoramidityl, or glycidyl. In a more preferred embodiment, R¹⁶ isactivated acyl. Alternatively, R¹⁶ is activated ester. In a still morepreferred embodiment, R¹⁶ is succinimidyloxy-ester orsulfosuccinimidyloxy-ester.

The compound has a balanced charge. In a preferred aspect, thecompound's net anionic charge is balanced by alkali metal counterions(e.g., sodium or potassium). In a more preferred aspect, at least one ofthe counterions is sodium. Alternatively, all of the counterions aresodium.

In an alternative embodiment, the compound has a balanced charge inwhich positively and negatively charged substituents are balanced sothat the dye molecule has a net charge of −1, 0, or +1 (preferably, 0),even without its counterions (i.e., the dye counterion has a net chargeof −1, 0, or +1). In some aspects, this net charge is produced byincluding numbers of positively and negatively charged substituentgroups that produce a dye net charge of −1, 0, or +1. This type ofcharge balancing is discussed in U.S. Provisional Application 61/150,522(filed Feb. 9, 2009) and WO 2010/091243 (filed Feb. 5, 2010), which areincorporated by reference.

In a preferred aspect, Q is

For example, the compounds set forth in Formula I comprise saidpreferred aspect of Q. More preferably, each R³, R^(4a), R^(4b), R^(5a),R^(5b), R^(6a), and R^(6b) are a member independently selected from thegroup of hydrogen, halo, and sulfonato.

In a preferred aspect, Q is

For example, the compounds set forth in Formula I comprise saidpreferred aspect of Q. More preferably, each R³, R^(4a), R^(4b), R^(5a),R^(5b), R^(6a), and R^(6b) are a member independently selected from thegroup of hydrogen, halo, and sulfonato.

In a more preferred aspect, the compound has the formula:

wherein M is a cationic counterion. More preferably, M is an alkalimetal ion.

In an alternative aspect, the compound has the formula:

In another alternative aspect, the compound has the formula:

wherein M is a cationic counterion. More preferably, M is an alkalimetal ion.

In another preferred aspect, the compound has the formula:

Alternatively, the compound has the formula:

Alternatively, the compound has the formula:

In certain aspects, an activated acyl group is present in place of thecarboxy group. In a still more preferred aspect, the activated acylgroup is an activated ester. In a still yet more preferred aspect, theactivated ester is a succinimidyloxy- ester.

In a first aspect, the compound of Formula I, Ia, II, or IIa has afluorescence absorption maximum at a wavelength within the range ofabout 550 nm to about 1000 nm. Preferably, the compound has afluorescence absorption maximum at a wavelength within the range ofabout 600 nm to about 1000 nm. More preferably, the compound has afluorescence absorption maximum at a wavelength within the range ofabout 600 nm to about 850 nm. Still more preferably, the compound has afluorescence absorption maximum at a wavelength within the range ofabout 600 nm to about 725 nm. Alternatively, the compound has afluorescence absorption maximum at a wavelength within the range ofabout 725 nm to about 850 nm.

The present application broadly encompasses all possible stereoisomersof the compounds as described herein, including the variousdiasteromers, enantiomers, and olefin stereoisomers apparent to one ofskill in the art. This application is further directed to all methods ofpurifying cyanine dye compound stereoisomers that are well-known in theart as well as the purified compounds available by these methods.

Preparation of Compounds of Formula I

In one aspect, the preferred cyanine compounds set forth in Formula Iare prepared by means of an organometallic coupling to incorporate asubstituent to the polymethine bridge. More preferably, the substituentis installed by means of a palladium coupling. The substituent canoptionally be modified after its inclusion (e.g., deprotected, activatedfor reaction with a biomolecule, or reacted to form a linking group).

The Miyaura-Suzuki reaction, also known as the Suzuki coupling, has beenextensively used in organic synthesis since its discovery: Miyaura, N.;Yamada, K.; Suzuki, A. Tetrahedron Lett. 1979, 36, 3437-3440. Recently aSuzuki coupling was used to install a substituted aryl substituent atthe central position of a heptamethine bridge in a water-soluble cyaninedye: Lee, H.; Mason, J. C.; Achilefu, S. J. Org. Chem. 2006, 71,7862-7865.

However, because many cyanine dyes decompose under standard Suzukicoupling conditions of heating with a base, few examples of its use forthe synthesis of cyanine dyes are known.

In a particularly preferred aspect of the instant invention, thesubstituent of a compound of Formula I is incorporated by means of aSuzuki coupling reaction, some of which are detailed in the examples ofthis specification. In one embodiment, the polymethine substrate for theSuzuki coupling is a 3-halopentamethine or a 4-haloheptamethine. In apreferred embodiment, the halo- substituent is a chloride or a bromide.In a more preferred embodiment, the halo- substituent is a bromide.

Other means of preparing cyanine dyes and their synthetic precursors areincluded in Hamer, F. M., Cyanine Dyes and Related Compounds,Weissberger, Mass., ed. Wiley Interscience, N.Y. 1964; and Mojzych, M.,Henary, M. “Synthesis of Cyanine Dyes,” Top. Heterocycl. Chem., vol. 14,Springer Berlin, Heildelberg, 2008, pp. 1-9. Further, U.S. Pat. Nos.4,337,063; 4,404,289; and 4,405,711 describe a synthesis for a varietyof cyanine dyes having N-hydroxysuccinimide active ester groups. U.S.Pat. No. 4,981,977 describes a synthesis for cyanine dyes havingcarboxylic acid groups. U.S. Pat. No. 5,268,486 discloses a method formaking arylsulfonate cyanine dyes. U.S. Pat. No. 6,027,709 disclosesmethods for making cyanine dyes having phosphoramidite groups. U.S. Pat.No. 6,048,982 discloses methods for making cyanine dyes having areactive group selected from the group of isothiocyanate, isocyanate,phosphoramidite, monochlorotriazine, dichlorotriazine, mono- ordi-halogen substituted pyridine, mono- or di-halogen substituteddiazine, aziridine, sulfonyl halide, acid halide, hydroxysuccinimideester, hydroxy sulfosuccinimide ester, imido ester, glyoxal andaldehyde.

One common synthetic route involves preparing substituted orunsubstituted indolesulfonate quaternary salts according to proceduresthat are well-known in the art, some of which are detailed in theexamples of this specification. Particularly preferred indole quaternarysalts include, among others, indolesulfonate and benzindolesulfonatequaternary salts, which are exemplified in this specification.

The pair of synthesized salts are then reacted with a dialdehyde or adialdehyde equivalent (e.g., a Schiff base) to form the polymethinebridge by means of techniques and reaction conditions that arewell-known in the art, some of which are detailed in the examples ofthis specification. Preferably, one of the dialdehydes is protected ormasked to allow incorporation of one polycyclic side of the bridge(e.g., the indoline ring), followed by deprotection or unmasking of thealdehyde and by incorporation or construction of the other polycyclicgroup (e.g., the pyrrolopyridine). Schiff bases can be purchased fromcommercial suppliers (e.g., Sigma-Aldrich) or prepared according toprocedures that are well-known in the art (e.g., the method of Example5).

Methods of Labeling Biomolecules

The cyanine compounds of Formula I can be attached to biomolecules,which are defined above. Methods of linking dyes to various types ofbiomolecules are well-known in the art. For a thorough review of, e.g.,oligonucleotide labeling procedures, see R. Haugland in Excited Statesof Biopolymers, Steiner ed., Plenum Press (1983), Fluorogenic ProbeDesign and Synthesis: A Technical Guide, PE Applied Biosystems (1996),and G. T. Herman, Bioconjugate Techniques, Academic Press (1996).

“Click” chemistry provides one possible way for linking the inventivedyes to biomolecules. Click chemistry uses simple, robust reactions,such as the copper-catalyzed cycloaddition of azides and alkynes, tocreate intermolecular linkages. For a review of click chemistry, seeKolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem. 2001, 40, 2004,

Connection (or ligation) of two fragments to make a larger molecule orstructure is often achieved with the help of so-called “click chemistry”described by Sharpless et al. Angew. Chem, Int. Ed. 40: 2004 (2001).This term is used to describe a set of bimolecular reactions between twodifferent reactants such as azides and acetylenes. The formation of1,2,3-triazoles in 1,3-dipolar cycloaddition of azides to a triple bondis known, but because the activation energy of acetylene-azidecycloaddition is relatively high, the reaction is slow under ambientconditions.

The utility of the reaction of azides with alkynes was expanded by thediscovery of Cu (I) catalysis. 1,3-cycloaddition of azides to terminalacetylenes in the presence of catalytic amounts of cuprous salts isfacile at room temperature in organic or aqueous solutions.

U.S. Pat. No. 7,807,619 to Bertozzi et al. teaches modified cycloalkynecompounds and method of use of such compounds in modifying biomolecules.Bertozzi et al. teach a cycloaddition reaction that can be carried outunder physiological conditions. As disclosed therein, a modifiedcycloalkyne is reacted with an azide moiety on a target biomolecule,generating a covalently modified biomolecule.

The present invention provides cyanine dyes with click chemistryfunctionalities useful for labeling biomolecules. As such, in oneaspect, the present invention provides compounds of Formula I or II, inwhich I one embodiment, each R¹⁶ is independently a member selected fromthe group consisting of activated acyl, acrylamido, optionallysubstituted alkylsulfonate ester, azido, optionally substitutedarylsulfonate ester, amino, azido, aziridino, boronato, diazo, formyl,glycidyl, halo, haloacetamidyl, haloalkyl, haloplatinato, halotriazino,hydrazinyl, imido ester, isocyanato, isothiocyanato, maleimidyl,mercapto, phosphoramidityl, a photoactivatable moiety, vinyl sulfonyl,alkynyl, a pegylated azido group, and a pegylated alkynyl group; and inwhich at least one R¹⁶ is independently a member selected from the groupazido, alkynyl, a pegylated azido and a pegylated alkynyl.

In yet other aspects, the present invention relates to two componentsthat interact with each other to form a stable covalent bio-orthogonalbond. Bio-orthogonal reactions are reactions of materials with eachother, wherein each material has limited or essentially no reactivitywith functional groups found in vivo. These components are of use inchemical and biological assays, as chemical reagents, medical imagingand therapy, and more particularly, in nucleic acid modificationtechniques. According to a particular embodiment of the invention, thecovalent bio-orthogonal bond is obtained by the [3+2] cycloaddition ofazides and alkynes.

In still other aspects, one of the two components that interact witheach other to form a stable covalent bio-orthogonal bond is a nearinfrared dye, such as a cyanine dye. In a preferred aspect, the cyaninedyes of the present invention comprise either an azide or an alkynegroup for use as a reactant in a click chemistry reaction and the otherreactant is a biomolecule such as a nucleotide comprising either analkyne or azide group.

Azide reactive groups such as an alkyne compounds can react with atleast one 1,3-dipole-functional compound such as an alkyne reactivegroup (e.g., a azido group) in a cyclization reaction to form aheterocyclic compound. In certain embodiments, the reaction can becarried out in the presence of an added catalyst (e.g., Cu(I)). In otherembodiments, the reaction is carried out in the absence of suchcatalysts. Exemplary 1,3-dipole-functional compounds include, but arenot limited to, azide-functional compounds, nitrile oxide-functionalcompounds, nitrone-functional compounds, azoxy-functional compounds,and/or acyl diazo-functional compounds. Preferably, azide-functionalcompounds are used.

Suitable biomolecule moieties for click reaction include, for example,monomeric and polymeric derivatives of nucleotides, carbohydrates, aminoacids, lipids, glycols, alkanes, alkenes, arene, silicates, as well asbiologically active and inactive compounds obtained from nature or fromartificial synthesis.

Other suitable biological molecules include those having a azido oralkynyl functionality, which include, but are not limited to, anantibody, an antigen, an avidin, a carbohydrate, a deoxy nucleic acid, adideoxy nucleotide triphosphate, an enzyme cofactor, an enzymesubstrate, a fragment of DNA, a fragment of RNA, a hapten, a hormone, anucleic acid, a nucleotide, a nucleotide triphosphate, a nucleotidephosphate, a nucleotide polyphosphate, an oligosaccharide, a peptide,PNA, a polysaccharide, a protein, a streptavidin, and the like. Thesebiological molecules will in turn be reacted with the dye compounds ofthe present invention comprising either an azide or an alkyne group foruse in click chemistry reactions.

In one aspect, the cyanine compounds of Formula I have sufficientsolubility in aqueous solutions that once they are conjugated to asoluble ligand or biomolecule, the ligand or biomolecule retains itssolubility. In certain instances, the bioconjugates also have goodsolubility in organic media (e.g., DMSO or DMF), which providesconsiderable versatility in synthetic approaches to the labeling ofdesired materials.

In another aspect, the present invention provides a method or processfor labeling a ligand or biomolecule with a compound of Formula I, themethod comprising: contacting a ligand or biomolecule with a compoundhaving Formula I or Ia to generate the corresponding bioconjugatecompound of Formula II or IIa.

In one preferred embodiment, the R¹⁶ group or the R¹³ group reacts witha thiol, a hydroxyl, a carboxyl, or an amino group on a biomolecule,forming a linking group between the dye and the biomolecule. In a morepreferred embodiment, this reaction is carried out in mixtures ofaqueous buffer and an organic solvent such as DMF at pH 8 to 9.Alternatively, this reaction is carried out in distilled water or in anaqueous buffer solution. For thiols or for acidic groups, a pH of 7 orlower is preferred for the reaction solvent, especially if a substratealso contains a reactive amino group.

Selected examples of reactive functionalities useful for attaching acompound of Formula I to a ligand or biomolecule are shown in Table 1,wherein the bond results from the reaction of a dye with a ligand orbiomolecule. Column A of Table 1 is a list of the reactivefunctionalities, which can be on the compound of Formula I or thebiomolecule. Column B is a list of the complementary reactive groups(preferably, a carboxyl, hydroxyl, thiol, or amino functionality), whichcan be on the biomolecule or the compound of Formula I, and which reactwith the indicated functionality of Column A to form the bond of ColumnC. Those of skill in the art will know of other bonds suitable for usein the present invention.

TABLE 1 Exemplary Bonds for Linking Groups A B Reactive FunctionalityComplementary Group (Compound of Formula I (Biomolecule or C orBiomolecule) Compound of Formula I) Resulting Linking Group activatedesters* amines/anilines amides acrylamides thiols thioethers acylazides** amines/anilines amides acyl halides amines/anilines amides acylhalides alcohols/phenols esters acyl nitriles alcohols/phenols estersacyl nitriles amines/anilines amides aldehydes amines/anilines iminesaldehydes or ketones hydrazines hydrazones aldehydes or ketoneshydroxylamines oximes alkyl halides amines/anilines alkyl amines alkylhalides carboxylic acids esters alkyl halides thiols thioethers alkylhalides alcohols/phenols ethers anhydrides alcohols/phenols estersanhydrides amines/anilines amides/imides aryl halides thiols thiophenolsaryl halides amines aryl amines azides alkynes 1,2,3-triazoles azidesester with phosphine amide (and phosphine reagent (e.g., o- oxide)diphenylphosphino group) aziridines thiols thioethers boronates glycolsboronate esters boronates/boronic acids aryl halides C—C bond to arylring boronates/boronic acids alkenyl halides C—C bond to alkenyl groupactivated carboxylic acids amines/anilines amides activated carboxylicacids alcohols esters activated carboxylic acids hydrazines hydrazidescarbodiimides carboxylic acids N-acylureas or anhydrides diazoalkanescarboxylic acids esters electron-rich diene dienophile (e.g., electron-cyclohexene (Diels-Alder poor alkene) cycloaddition) epoxides thiolsthioethers epoxides amines alkyl amines epoxides carboxylic acids estershaloacetamides thiols thioethers haloplatinate amino platinum complexhaloplatinate heterocycle platinum complex halotriazines amines/anilinesaminotriazines halotriazines alcohols/phenols triazinyl ethers imidoesters amines/anilines amidines isocyanates amines/anilines ureasisocyanates alcohols/phenols urethanes isothiocyanates amines/anilinesthioureas maleimides thiols thioethers phosphoramidites alcoholsphosphite esters photoactivatable group varies; see definition varies;see definition quadricyclanes π-electrophile (e.g., Ni norbornenecycloaddition bis(dithiolene)) product silyl halides alcohols silylethers sulfonyl azides thiocarboxylic acids N-acyl sulfonamidessulfonate esters amines/anilines alkyl amines sulfonate esterscarboxylic acids esters sulfonate esters thiols thioethers sulfonateesters alcohols/phenols ethers sulfonyl halides amines/anilinessulfonamides 1,2,4,5-tetrazine alkene dihydropyradazine vinyl sulfonylthiols thioethers vinyl sulfonyl activated diene cyclohexenyl(Diels-Alder) *Activated esters, as understood in the art, generallyhave the formula —C(O)OM, where —OM is a leaving group (e.g.succinimidyloxy (—OC₄H₄NO₂), sulfosuccinimidyloxy (—OC₄H₃NO₂SO₃H),-1-oxybenzotriazolyl (—OC₆H₄N₃); 4-sulfo-2,3,5,6-tetrafluorophenyl; oran aryloxy group or aryloxy substituted one or more times by electronwithdrawing substituents such as nitro, fluoro, chloro, cyano, ortrifluoromethyl, or combinations thereof, used to form activated arylesters; or —C(O)OM is a carboxylic acid activated by a carbodiimide toform an anhydride or mixed anhydride —C(O)OC(O)R^(a) or—C(O)OC(NR^(a))NHR^(b), wherein R^(a) and R^(b) are membersindependently selected from the group consisting of C₁—C₆ alkyl, C₁—C₆perfluoroalkyl, C₁—C₆ alkoxy, cyclohexyl, 3-dimethylaminopropyl, orN-morpholinoethyl). **Acyl azides can also rearrange to isocyanates.

Some methods of forming linking groups include those taught in Slettenand Bertozzi, J. Am. Chem. Soc. electronic publication atdx.doi.org/10.1021/ja2072934; Devaraj and Weissleder, Acc. Chem. Res.electronic publication at dx.doi.org/10.1021/ar200037t; Krishnamoorthyand Begley, J. Am. Chem. Soc. electronic publication atdx.doi.org/10.1021/ja1034107; and the like.

When linking a compound of Formula I having a carboxylic acid with anamine-containing ligand or biomolecule, the carboxylic acid can first beconverted to a more reactive form, e.g, a N-hydroxy succinimide (NHS)ester or a mixed anhydride, by means of an activating reagent. Theamine-containing ligand or biomolecule is treated with the resultingactivated acyl to form an amide linkage. In a more preferred embodiment,this reaction is carried out in aqueous buffer at pH 8 to 9 with DMSO orDMF as an optional co-solvent. Alternatively, this reaction is carriedout in distilled water or in an aqueous buffer solution.

Similarly, the attachment of an isocyanate- or isothiocyanate-containingcompound of Formula I is analogous to the procedure for the carboxy dye,but no activation step is required. The amine-containing ligand orbiomolecule is treated directly with the activated acyl compound to forma urea or a thiourea linkage. In a more preferred embodiment, thereaction is carried out in aqueous buffer at pH 9 to 10 with DMSO or DMFas an optional co-solvent. Alternatively, this reaction is carried outin distilled water or in an aqueous buffer solution.

If the compound of Formula I or biomolecule has a reactive hydroxylgroup, it can be linked to a ligand or biomolecule by means ofphosphoramidite chemistry, which ultimately forms a phosphate linkagebetween the dye and the biomolecule. For examples of such labelingmethods, see U.S. Pat. No. 6,027,709, which discloses many preferredlinking groups, linking methods, and biomolecules that can be readilylabeled. In one embodiment, solid-phase synthesis is preferred, asdisclosed in U.S. Pat. No. 6,027,709.

In a preferred embodiment, the biomolecule is DNA or RNA. Use ofphosphoramidite chemistry allows labeling of a DNA or an RNA during thesynthesis process. The protected nucleotide is labeled while attached toa solid-phase support. The free 5′-OH group is reacted with thephosphoramidite and a tetrazole activator to form a phosphite linkagewhich subsequently is oxidized to phosphate. The labeled DNA or RNA isthen cleaved from the solid phase by means of ammonia or by anotherstandard procedure.

It is generally preferred to prepare a phosphoramidite of a cyanine dyeto label DNA molecules in a DNA synthesizer. It is also preferred toattach the dye to the 5′ end of a protected, support-bondedoligonucleotide through standard phosphoramidite chemistry. For a listof preferred label terminators for use in DNA sequencing, see U.S. Pat.No. 5,332,666.

In another preferred embodiment, the biomolecule is an antibody. It ispreferred that antibody labeling is carried out in a buffer optionallyincluding an organic co-solvent, under basic pH conditions, and at roomtemperature. It is also preferred that the labeled antibody be purifiedby dialysis or by gel permeation chromatography using equipment such asa SEPHADEX® G-50 column to remove any unconjugated compound of FormulaI. Those of skill in the art will know of other ways and means forpurification.

In still another preferred embodiment, the biomolecule contains a thiolgroup that forms the linking group by reaction with a maleimidylsubstituent at R¹⁶. In a more preferred embodiment, the biomolecule is aprotein, a peptide, an antibody, a thiolated nucleotide, or a thiolateddeoxynucleotide.

In yet other aspects, the linking group or biomolecule comprises apolymer. In a preferred embodiment, the polymer is a member selectedfrom the group of a PEG, a copolymer of PEG-polyurethane, and acopolymer of PEG-polypropylene. In still yet other aspects, the linkinggroup is a member selected from the group of a polysaccharide, apolypeptide, an oligosaccharide, a polymer, a co-polymer and anoligonucleotide.

In one aspect, biomolecules can be labeled according to the presentinvention by means of a kit. In certain instances, the kit comprises abuffer and a dye as disclosed in the instant application (i.e., acompound of Formula I or Formula Ia). Preferably, the kit contains acoupling buffer such as 1 M KH₂PO₄ (pH 5), optionally with added acid orbase to modify the pH (e.g., pH 8.5 is preferred for reactions withsuccinimide esters and pH 7 is preferred for reactions with maleimides).Preferably, the buffer has a qualified low fluorescence background.

Optionally, the kit can contain a purification sub-kit. After labeling abiomolecule with a preferred dye, the labeled biomolecule may beseparated from any side reaction products and any free hydrolyzedproduct resulting from normal hydrolysis. For biomolecules containing 13or fewer amino acids, preparative thin layer chromatography (TLC) canremove impurities. In certain instances, preparative TLC, optionallyperformed with commercially available TLC kits, can be used to purifydye-labeled peptides or proteins.

For larger biomolecules such as larger peptides or proteins, a SEPHADEX®G-15, G-25, or G-50 resin may remove unwanted derivatives. In certaininstances, a Gel Filtration of Proteins Kit, which is commerciallyavailable from Life Sciences, can be used to separate dye-labeledpeptides and proteins from free dye. The labeled biomolecules thatremain after desalting can often be used successfully without furtherpurification. In some cases, it may be necessary to resolve and assessthe activity of the different products by means of HPLC or otherchromatographic techniques.

Bioconjugate Compounds

In another embodiment of the invention, a bioconjugate of the Formula IIis provided:

wherein Q^(L) is a member selected from the group of aone-polymethine-carbon segment and a three-polymethine-carbon segment:

respectively; wherein the segment is the central portion of either afive- or a seven-polymethine-carbon polymethine bridge.

In a preferred aspect, Q^(L) is a portion of a polymethine bridge thatis a pentamethine:

More preferably, Q^(L) is

In a second preferred aspect, Q^(L) is a portion of a polymethine bridgethat is a heptamethine:

More preferably, Q^(L) is

In an alternative preferred aspect, Q^(L) is a portion of a polymethinebridge that is a substituted heptamethine:

More preferably, Q^(L) is

In an alternative, more preferred aspect, the substituted heptamethineincludes a cycloalkyl ring:

Still more preferably, Q^(L) is

In a third preferred aspect, Q^(L) is selected from the group consistingof:

More preferably, Q^(L) is

In a fourth preferred aspect, Q^(L) is selected from the groupconsisting of:

R¹, R^(2a), R^(2b), R³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), R^(6b),R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, L, and Y are as previouslydefined for the compound of Formula I, including all preferredembodiments that are identified herein.

Each Z is independently selected from the group consisting of -L-R¹³ and-L-R^(L). In a more preferred aspect, Z is -L-R^(L), wherein L is abond.

Each R^(L) comprises 1) a linking group that connects the cyanine dyecompound to a biomolecule; and 2) the biomolecule to which it isconnected (i.e., the linking group and the biomolecule connectedthereby), wherein the compound comprises at least one R^(L). Preferredlinking groups are indicated in Table 1 (column C). In a particularlypreferred aspect, the linking group is an amide or an ester. In a moreparticularly preferred aspect, the linking group is an amide.

The compound has a balanced charge. In a preferred aspect, thecompound's net anionic charge is balanced by alkali metal counterions(e.g., sodium or potassium). In a more preferred aspect, at least one ofthe counterions is sodium. Alternatively, all of the counterions aresodium.

In another preferred embodiment of the bioconjugate, any preferredembodiments or aspects of the inventive compound of Formulas I and Iacan included in the embodiment of a bioconjugate. Representativeexamples of preferred compounds of Formulas I and Ia that correspond topreferred bioconjugate embodiments are described in the dependent claimsof the instant application.

A more preferred aspect of the bioconjugate has the following structure:

wherein M is a cationic counterion.

Another preferred aspect of the bioconjugate has the followingstructure:

wherein M is a cationic counterion.

More preferably, the compound has the following structure:

Alternatively, the bioconjugate has the following structure:

wherein M is a cationic counterion.

In certain aspects, a preferred biomolecule for the instant invention isselected from the group containing an acyclo terminator triphosphate, anantibody, an antigen, an avidin, a carbohydrate, a deoxy nucleic acid, adideoxy nucleotide triphosphate, an enzyme cofactor, an enzymesubstrate, a fragment of DNA, a fragment of RNA, a hapten, a hormone, anucleic acid, a nucleotide, a nucleotide triphosphate, a nucleotidephosphate, a nucleotide polyphosphate, an oligosaccharide, a peptide,PNA, a polysaccharide, a protein, a streptavidin, and the like.

Suitable nucleotides include nucleoside polyphosphates, including, butnot limited to, deoxyribonucleoside polyphosphates, ribonucleosidepolyphosphates, dideoxynucleoside polyphosphates, carbocyclic nucleosidepolyphosphates and acyclic nucleoside polyphosphates and analogsthereof. Suitable nucleotides also include nucleotides containing 3, 4,5, 6, or more phosphate groups, in the polyphosphate chain, where thephosphate (e.g., α, β, γ, ε, or terminal phosphate), sugar, base, orcombination thereof is labeled with a compound of Formula I. Thepolyphosphate nucleotides include, but are not limited to,tetraphosphates, pentaphosphates, hexaphosphates, heptaphosphates, andthe like. The bases include for example, purines, (adenine and guanine)pyrimidines, (thymine, uracil and cytosine) and derivatives thereof.

In certain instances, the dye of Formula I is attached to the phosphate(e.g. α, β, γ, ε-phosphate or terminal phosphate) through aphosphorothioate linkage (see, for example, U.S. Pat. No. 6,323,186,incorporated herein by reference), heteroatom, or functional group A, orB, resulting in linkage C of Table I. See also U.S. Pat. No. 6,399,335(incorporated herein by reference) entitled “γ-phosphoester nucleosidetriphosphates,” which provides methods and compositions for polymerizingparticular nucleotides with a polymerase using γ-phosphoester linkednucleoside triphosphates. Other ways of linking the compounds of FormulaI to a nucleotide are known to those of skill in the art. Using thesenucleotides with a DNA polymerase can lead to identification of specificnucleotides in a DNA or RNA sequence by identification of the labeledpyrophosphate or polyphosphate released upon incorporation of thenucleotide base into RNA or DNA. (See for example, U.S. Pat. No.6,232,075, US Pub. No. 2004/0241716 and U.S. Pat. No. 7,452,698 each ofwhich is incorporated herein by reference).

More preferred aspects include an antibody, an avidin, and astreptavidin. Even more preferred aspects include a goat anti-mouse(GAM) antibody, a goat anti-rabbit (GAR) antibody, and streptavidin.

In certain other aspects, preferred biomolecules for the instantinvention include somatostatin, endostatin, a carbohydrate, anoligosaccharide, an aptamer, a liposome, PEG, an angiopoietin,angiostatin, angiotensin II, α₂-antiplasmin, annexin V, β-cyclodextrintetradecasulfate, endoglin, endosialin, endostatin, epidermal growthfactor, fibrin, fibrinopeptide β, fibroblast growth factor, FGF-3, basicfibronectin, fumagillin, heparin, hepatocycle growth factor, hyaluronan,an insulin-like growth factor, an interferon-α, β inhibitor, ILinhibitor, laminin, leukemia inhibitory factor, linomide, ametalloproteinase, a metalloproteinase inhibitor, an antibody, anantibody fragment, an acyclic RGD peptide, a cyclic RGD peptide,placental growth factor, placental proliferin-related protein,plasminogen, plasminogen activator, plasminogen activator inhibitor-1, aplatelet activating factor antagonist, platelet-derived growth factor, aplatelet-derived growth factor receptor, a platelet-derived growthfactor receptor, platelet-derived endothelial cell growth factor,pleiotropin, proliferin, proliferin-related protein, a selectin, SPARC,a snake venom, substance P, suramin, a tissue inhibitor of ametalloproteinase, thalidomide, thrombin, thrombin-receptor-activatingtetradecapeptide, transformin growth factor-α, β, transforming growthfactor receptor, tumor growth factor-α, tumor necrosis factor,vitronectin, and the like.

In still other aspects, preferred biomolecules include a carbohydrateand a carbohydrate derivative. Representative examples includeglucosamine, a glyceraldehyde, erythrose, threose, ribose, arabinose,xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose,galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose,sorbose, tagatose, and a derivative thereof. Even more preferredbiomolecules include 2-deoxy-D-glucose, 2-deoxy-L-glucose, and racemic2-deoxyglucose.

In yet still other aspects, the biomolecule can be a ligand that hasaffinity for a receptor selected from the group of EGFR, Her2, PDGFR,IGFR, c-Ryk, c-Kit, CD24, integrins, FGFR, KFGR, VEGFR, TRAIL decoyreceptors, retinoid receptor, growth receptor, PPAR, vitamin receptor,glucocordicosteroid receptor, Retinoid-X receptor, RHAMM, high affinityfolate receptors, Met receptor, estrogen receptor and Ki67. Preferably,the biomolecule is a ligand that has affinity for an integrin receptor.

Alternatively, the biomolecule is selected from the group ofsomatostatin, endostatin, a carbohydrate, a monosaccaride, adisaccharide, a trisaccharide, an oligosaccharide, aptamer, liposome andpolyethylene glycol.

In yet another aspect, the biomolecule is a small-molecule drug ordrug-like molecule such as a tetracycline antibiotic, a tetracyclinederivative, and calcein.

Alternatively, the biomolecule is a small-molecule drug or peptide.

In other aspects, a cyanine dye set forth in an embodiment of thepresent invention is conjugated to a biological cell. Preferably, thedye is conjugated by means of an R^(L) linking group.

In other aspects, a preferred biomolecule for the instant invention isselected from the group containing an antigen and a hapten. Preferably,the biomolecule is an immunogen.

In other aspects, a preferred biomolecule for the instant invention isselected from the group containing an enzyme cofactor and an enzymesubstrate.

In other aspects, a preferred biomolecule for the instant invention isselected from the group containing an amino acid, a carbohydrate, ahapten, a hormone, a glycoprotein, a liposome, a nucleic acid, anucleotide, a nucleotide triphosphate, a nucleotide polyphosphate, anoligosaccharide, a peptide, a peptide nucleic acid, a polyalkyleneglycol, a polysaccharide, a protein, a small-molecule drug, and a snakevenom.

More preferably, the preferred biomolecule is selected from the groupcontaining angiostatin, endostatin, fumagillin, a fumagillin derivative,placental proliferin-related protein, plasminogen, somatostatin, andthalidomide.

Alternatively, the biomolecule is an aptamer.

Alternatively, the biomolecule is an a small-molecule drug. Preferably,the drug is selected from the group of tetracyclin, a tetracyclinantibiotic, and a derivative thereof.

Alternatively, the biomolecule is an integrin.

Alternatively, the biomolecules is selected from the group containing anantibody and an antibody fragment.

Alternatively, the biomolecule is selected from the group containingpolyethylene glycol.

Alternatively, the biomolecule is selected from the group containing anangiopoietin, epidermal growth factor, a fibroblast growth factor,hepatocyte growth factor, an insulin-like growth factor, placentalgrowth factor, platelet-derived growth factor, a platelet-derived growthfactor receptor, a platelet-derived endothelial cell growth factor,transforming growth factor-α, transforming growth factor-β, andtransforming growth factor receptor. More preferably, the fibroblastgrowth factor is fibroblast growth factor 3.

Alternatively, the biomolecule is selected from the group containing anacyclic RGD peptide, a cyclic RGD peptide, endosialin, and a derivativethereof. Preferably, the biomolecule is an acyclic RGD peptide, a cyclicRGD peptide, or a derivative thereof. More preferably, the cyclic RGDpeptide is cyclo (Arg-Gly-Asp-D-Phe-Lys) (i.e., c(RGDfK)).

Alternatively, the biomolecule is selected from the group containingα₂-antiplasmin, plasminogen, plasminogen activator, plasminogenactivator inhibitor-1, and plasminogen activator inhibitor-2.

Alternatively, the biomolecule is selected from the group containingfibrin, fibrinopeptide β, thrombin, and thrombin-receptor-activatingtetradecapeptide.

Alternatively, the biomolecule is selected from the group containing anacyclo terminator triphosphate, a deoxynucleic acid, a ribonucleic acid,a nucleotide, a nucleotide triphosphate, a nucleotide polyphosphate, anda peptide nucleic acid.

Alternatively, the biomolecule is selected from the group containing afragment of RNA and a fragment of DNA.

Alternatively, the biomolecule is selected from the group containingangiotensin II and substance P.

Alternatively, the biomolecule is selected from the group containing alectin and a selectin.

Alternatively, the biomolecule is selected from the group containingendoglin, a laminin, a fibronectin, SPARC, and vitronectin.

Alternatively, the biomolecule is selected from the group containing ametalloproteinase and a metalloproteinase inhibitor.

Alternatively, the biomolecule is a tissue inhibitor of ametalloproteinase.

Alternatively, the biomolecule is a platelet activating factorantagonist.

Alternatively, the biomolecule is selected from the group containingβ-cyclodextrin tetradecasulfate, heparin, hyaluronan, and a derivativethereof. Preferably, the biomolecule is selected from the groupconsisting of hyaluronan and a derivative thereof.

Alternatively, the biomolecule is an annexin.

Alternatively, the biomolecule is selected from the group containinginterleukin inhibitor, leukemia inhibitory factor, pleiotropin, andtumor necrosis factor. More preferably, the biomolecule is aninterleukin-1 receptor antagonist.

Alternatively, the biomolecule is selected from the group containingproliferin and a proliferin-related protein.

Alternatively, the biomolecule is selected from the group containingcalcein, laquinimod, linomide, and suramin.

Alternatively, the biomolecule is an interferon-α,β inhibitor.

Alternatively, the biomolecule is selected from the group containingtyramine and a tyramine derivative.

Alternatively, the biomolecule is selected from the group containing anavidin, biotin, and a streptavidin.

Alternatively, the biomolecule is selected from the group containing aglucosamine, a glyceraldehyde, erythrose, threose, ribose, arabinose,xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose,galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose,sorbose, tagatose, and a derivative thereof. More preferably, thebiomolecules include 2-deoxy-D-glucose, 2-deoxy-L-glucose, and racemic2-deoxyglucose.

Methods of Imaging

In another embodiment, the compounds of Formula I or Ia can be used asin vitro or in vivo optical imaging agents of tissues and organs invarious biomedical applications. In one embodiment, the presentinvention provides a method for imaging, the method comprisingadministering a compound of Formula I or Ia.

In certain preferred aspects of the invention, any of the embodiments oraspects of the inventive compound of Formula I or Ia that are describedherein can be used in the method of imaging. Representative examples ofpreferred compounds for use in the method are described in thespecification and the dependent claims of the instant application.

In another embodiment, the present invention provides a method forimaging, the method comprising administering a compound of Formula II orIIa.

In certain preferred aspects of the invention, any of the embodiments oraspects of the inventive compound of Formula II or IIa that aredescribed herein can be used in the method of imaging. Representativeexamples of preferred compounds for use in the method are described inthe specification and the dependent claims of the instant application.

In certain preferred aspects, the compounds of the present invention areused as in vivo imaging agents of tissues and organs in variousbiomedical applications including, but not limited to, tomographicimaging of organs, monitoring of organ functions, coronary angiography,fluorescence endoscopy, imaging of tumors, laser guided surgery,photoacoustic and sonofluorescence methods, and the like. In one aspect,the compounds of the invention are useful for the detection of thepresence of tumors and other abnormalities by monitoring the bloodclearance profile of the dyes. In another aspect of the invention, thecompounds are useful for laser assisted guided surgery for the detectionof micro-metastases of tumors upon laparoscopy. In yet another aspect,the compounds are useful in the diagnosis of atherosclerotic plaques andblood clots.

In further aspects, the compounds of the present invention are used inthe imaging of: (1) ocular diseases in ophthalmology, for example, toenhance visualization of chorioretinal diseases, such as vasculardisorders, retinopathies, neovascularization, and tumors via directmicroscopic imaging; (2) skin diseases such as skin tumors via directmicroscopic imaging; (3) gastrointestinal, oral, bronchial, cervical,and urinary diseases and tumors via endoscopy; (4) atheroscleroticplaques and other vascular abnormalities via flexible endoscopiccatheters; (5) breast tumors via 2D- or 3D-image reconstruction; and (6)brain tumors, perfusion, and stroke via 2D- or 3D-image reconstruction.

In certain aspects, the compounds of the invention that arebioconjugates are particularly useful for imaging tumors, tissues, andorgans in a subject. For example, the existence of cancer cells orcancer tissues can be verified by labeling an anti-tumor antibody with acompound of Formula I and then administering the bioconjugated antibodyto the subject for detection and imaging of the tumor. Conjugatesbetween the dye compound and other antibodies, peptides, polypeptides,proteins, ligands for cell surface receptors, small molecules, and thelike are also useful agents for the in vivo imaging of tumors, tissues,and organs in a subject.

In certain aspects, the compounds of the invention may be administeredeither systemically or locally to the organ or tissue to be imaged,prior to the imaging procedure. In one aspect, the compounds areadministered intravenously. In another aspect, the compounds areadministered parenterally. In yet another aspect, the compounds areadministered enterally. The compositions used for administration of thecompound typically contain an effective amount of the compound orconjugate along with conventional pharmaceutical carriers and excipientsappropriate for the type of administration contemplated. For example,parenteral formulations advantageously contain a sterile aqueoussolution or suspension of a compound of Formula I (or Ia) or abioconjugate of Formula II (or IIa). Compositions for enteraladministration typically contain an effective amount of the compound orbioconjugate in aqueous solution or suspension that may optionallyinclude buffers, surfactants, thixotropic agents, flavoring agents, andthe like.

In certain aspects, the compositions are administered in doses effectiveto achieve the desired optical image of a tumor, tissue, or organ. Suchdoses may vary widely, depending upon the particular compound orbioconjugate employed, the tumor, tissue, or organ subjected to theimaging procedure, the imaging equipment being used, and the like.

In an alternative aspect, the method of the present invention providesfor administering to the subject a therapeutically effective amount of acompound; a targeting agent, such as a bioconjugate; or mixturesthereof. In one aspect, the targeting agent selectively binds to thetarget tissue. Light at a wavelength or waveband corresponding to thatwhich is absorbed by the photosensitizing agent is then administered. Inanother aspect, the compounds of the present invention act agentscapable of binding to one or more types of target cells or tissues, whenexposed to light of an appropriate waveband, absorb the light, causingsubstances to be produced that illuminate, impair or destroy the targetcells or tissues. Preferably, the compound is nontoxic to the subject towhich it is administered or is capable of being formulated in a nontoxiccomposition that can be administered to the subject. In addition,following exposure to light, the compound in any resulting photodegradedform is also preferably nontoxic.

In yet another aspect, the compounds of the present invention areadministered by any means known in the art, including, but not limitedto, ingestion, injection, transcutaneous administration, transdermaladministration, intravenously, subcutaneously and the like. Preferably,the compounds are administered transcutaneously, intravenously,subcutaneously, or intramuscularly to a subject.

In certain aspects, during imaging, the light passes through unbrokentissue. Where the tissue layer is skin or dermis, such transcutaneousimaging includes transdermal imaging, and it will be understood that thelight source is external to the outer skin layer. In some aspects (i.e.,transillumination), the light passes through a tissue layer, such as theouter surface layer of an organ (e.g., the liver). In such cases, thelight source is preferably external to the organ, but internal orimplanted within the subject or patient.

In further aspects of the invention, the target tumor, tissue, or organfor treatment is selected from the group of vascular endothelial tissue,an abnormal vascular wall of a tumor, a solid tumor, a tumor of thehead, a tumor of the neck, a tumor of a the gastrointestinal tract, atumor of the liver, a tumor of the breast, a tumor of the prostate, atumor of the ovary, a tumor of the uterus, a tumor of the testicle, atumor of the lung, a nonsolid tumor, malignant cells of one of ahematopoietic tissue and a lymphoid tissue, lesions in the vascularsystem, a diseased bone marrow, neuronal tissue or diseased neuronaltissue, and diseased cells in which the disease is one of an autoimmuneand an inflammatory disease. In yet a further aspect, the target tissueis a lesion in the vascular system of a type selected from the group ofatherosclerotic lesions, arteriovenous malformations, aneurysms, andvenous lesions.

In still further aspects, the forms of energy include, but are notlimited to, light (i.e., radiation), thermal, sonic, ultrasonic,chemical, light, microwave, ionizing (such as x-ray and gamma ray),mechanical, and electrical. The term “radiation” as used herein includesall wavelengths and wavebands. Preferably, the radiation wavelength orwaveband is selected to correspond with or at least overlap thewavelengths or wavebands that excite the photosensitizing agent.Compounds of the instant invention typically have one or more absorptionwavebands that excite them to produce the substances which illuminate,damage or destroy target cells, tissues, organs, or tumors. Preferably,the radiation wavelength or waveband matches the excitation wavelengthor waveband of the photosensitizing agent and has low absorption by thenon-target cells and the rest of the subject, including blood proteins.More preferably, the radiation wavelength or waveband is within the NIRrange of about 600 nm to about 1000 nm or a related range thereof (e.g.,the ranges that are described in the instant claims).

In certain aspects, the compounds of the present invention are used todirectly stain or label a sample so that the sample can be identified orquantitated. For instance, such compounds can be added as part of anassay for a biological target analyte, as a detectable tracer element ina biological or non-biological fluid; or for such purposes asphotodynamic therapy of tumors, in which a dyed sample is irradiated toselectively destroy tumor cells and tissues; or to photoablate arterialplaque or cells, usually through the photosensitized production ofsinglet oxygen.

Typically, the sample is obtained directly from a liquid source or as awash from a solid material (organic or inorganic) or a growth medium inwhich cells have been introduced for culturing, or a buffer solution inwhich cells have been placed for evaluation. Where the sample comprisescells, the cells are optionally single cells, including microorganisms,or multiple cells associated with other cells in two or threedimensional layers, including multicellular organisms, embryos, tissues,biopsies, filaments, biofilms, and the like.

A detectable optical response as used herein includes a change in, oroccurrence of, an optical signal that is detectable either byobservation or instrumentally. Typically the detectable response is achange in fluorescence, such as a change in the intensity, excitation oremission wavelength distribution of fluorescence, fluorescence lifetime,fluorescence polarization, or a combination thereof. The degree and/orlocation of staining, compared with a standard or expected response,indicates whether and to what degree the sample possesses a givencharacteristic. Some compounds of the invention may exhibit littlefluorescence emission, but are still useful as quenchers or chromophoricdyes. Such chromophores are useful as energy acceptors in FRETapplications, or to simply impart the desired color to a sample orportion of a sample.

FRET is a process by which a donor molecule (e.g., a dye) absorbs light,entering an excited state. Rather than emitting light, the firstmolecule transfers its excited state to a acceptor molecule with otherproperties (e.g., a dye fluorescing at a different wavelength or aquencher), and the acceptor fluoresces or quenches the excitation.Because the efficiency of the transfer is dependant on the twomolecules' proximity, it can indicate information about molecularcomplex formation or biomolecular structure. It can also indicate wherea particular complex is located within a cell or organism (e.g., FREToptical microscopy). For ways to use similar dyes as acceptors(quenchers) in FRET processes, see X. Peng, H. Chen, D. R. Draney, W.Volcheck, A. Schultz-Geschwender, and D. M. Olive, “A nonfluorescent,broad-range quencher dye for Förster resonance energy transfer assays,”Anal. Biochem 2009, 388(2): 220-228.

In certain aspects, for biological applications, the compounds of theinvention are typically used in an aqueous, mostly aqueous, oraqueous-miscible solution prepared according to methods generally knownin the art. The exact concentration of compound is dependent upon theexperimental conditions and the desired results, but ranges of 0.00001mM up to 0.1 mM, such as about 0.001 mM to about 0.01 mM, are possible.The optimal concentration is determined by systematic variation untilsatisfactory results with minimal background fluorescence isaccomplished.

In certain aspects, the method may involve treatment of an animal orsample with a dose comprising a compound of Formula I, a bioconjugate ofFormula II, or any of the aspects or embodiments thereof. The exactconcentration of compound is dependent upon the subject and the desiredresults. In certain embodiments, a dose of at least about 0.001, 0.005,0.01, 0.025, 0.05, or 0.075 mg/kg is used. Alternatively, a dose of atmost about 0.001, 0.005, 0.01, 0.025, 0.05, or 0.075 mg/kg is used. Incertain other embodiments, a dose of at least about 0.1, 0.25, 0.5, or0.75 mg/kg is used. Alternatively, a dose of at most about 0.1, 0.25,0.5, or 0.75 mg/kg is used. In still other embodiments, a dose of atleast about 0.1, 0.25, 0.5, or 0.75 mg/kg is used. Alternatively, a doseof at most about 0.1, 0.25, 0.5, or 0.75 mg/kg is used. In yet stillother embodiments, a dose of at least about 1, 2.5, 5, or 7.5 mg/kg isused. Alternatively, a dose of at most about 1, 2.5, 5, or 7.5 mg/kg isused. In additional other embodiments, a dose of at least about 10, 25,50, or 75 mg/kg is used. Alternatively, a dose of at most about 10, 25,50, or 75 mg/kg is used. In additional still other embodiments, a doseof at least about 100, 250, 500, or 750 mg/kg is used. Alternatively, adose of at most about 100, 250, 500, or 750 mg/kg is used. Other amountsfor administration of an effective dose may be readily determined by oneof skill in the art.

In certain aspects, in vitro, the compounds are advantageously used tostain samples with biological components. The sample can compriseheterogeneous mixtures of components (e.g., mixtures including intactcells, fixed cells, cell extracts, bacteria, viruses, organelles, andcombinations thereof), or a single component or homogeneous group ofcomponents (e.g. natural or synthetic amino acid, nucleic acid orcarbohydrate polymers, or lipid membrane complexes). Within theconcentrations of use, these compounds are generally non-toxic to livingcells and other biological components.

The compound is combined with the sample in any way that facilitatescontact between the compound and the sample components of interest.Typically, the compound or a solution containing the compound is simplyadded to the sample. Certain compounds of the invention, particularlythose that are substituted by one or more sulfonic acid moieties, tendto be impermeant to membranes of biological cells, and once insideviable cells, they are typically well-retained. Treatments thatpermeabilize the plasma membrane, such as electroporation, shocktreatments or high extracellular ATP, can be used to introduce selectedcompounds into cells. Alternatively, selected dye compounds can bephysically inserted into cells, e.g., by pressure microinjection, scrapeloading, patch clamp methods, or phagocytosis.

Alternatively, dye compounds can be conjugated to a biomolecule thatincreases their uptake into cells (e.g., cell-penetrating peptides suchas Tat, penetratin, transportin, derivatives thereof (e.g., Tatderivatives incorporating β- and γ-amino acids), and the like). Thisgeneral approach is usable in vitro or in vivo.

In certain aspects, at any time after or during staining, the sample isilluminated with a wavelength of light selected to give a detectableoptical response, and observed with a means for detecting the opticalresponse. Equipment that is useful for illuminating the compounds of theinvention includes, but is not limited to, hand-held ultraviolet lamps,mercury arc lamps, xenon lamps, lasers and laser diodes. Theseillumination sources are optionally integrated into laser scanners,fluorescence microplate readers, standard or minifluorometers, orchromatographic detectors. Preferred aspects of the invention arecompounds that are excitable at or near the wavelengths 633-636 nm, 647nm, 649 nm, 651 nm, 647-651 nm, 660 nm, 674 nm, 675 nm, 678 nm, 680 nm,674-680 nm, 685 nm, 674-685 nm, 680-685 nm, 685-690 nm, 690-695 nm,690-700 nm, and beyond 700 nm, such as 780 nm, 810 nm and 850 nm, asthese regions closely match the output of exemplary compounds or ofrelatively inexpensive excitation sources.

The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD cameras, video cameras,photographic film, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or by means foramplifying the signal such as photomultiplier tubes. Where the sample isexamined by means of a flow cytometer, examination of the sampleoptionally includes sorting portions of the sample according to theirfluorescence response.

EXAMPLES

Below, the present invention will be described by way of examples, whichare provided for illustrative purposes only. Accordingly, they are notto be construed as limiting the scope of the present invention asdefined by the appended claims.

Example 1 Preparation of5-Chloro-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine (1)

5-Chloro-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine (1)

To 2,5-dichloropyridine (40. g, 0.27 mol) in 2-methoxyethanol (200 mL)was added anhydrous hydrazine (20.0 mL, 0.625 mol). The mixture washeated at 110° C. for 3 h to generate the 5-chloro-hydrazinopyridine. Toform the hydrazone, a mixture of 10 g of the hydrazinopyridine and 11 mLof 3-methyl-2-butanone in 40 mL of benzene was heated at refluxovernight in a flask equipped with a Dean-Stark trap. All of thevolatile components were removed under reduced pressure, and theresulting hydrazone residue was heated in 50 g of polyphosphoric acid at135° C. for 45 min. The reaction mixture was poured into water,neutralized with sodium hydroxide, and extracted with ethyl acetate. Theresulting crude residue was purified by chromatography on silica gel(1:1 ethyl acetate/hexanes) to yield 2.50 g of5-chloro-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine (1).

Example 2 Preparation of 2,3,3-Trimethyl-3H-pyrrolo[2,3-b]pyridine (2)

2,3,3-Trimethyl-3H-pyrrolo[2,3-b]pyridine (2)

Compound 2 is prepared analogously to compound 1 (Example 1), exceptthat 2-hydrazinopyridine is used as a starting material.

Example 3 Preparation of5-Bromo-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine (3)

5-Bromo-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine (3)

Compound 3 was prepared analogously to compound 1 (Example 1), exceptthat 2,5-dibromopyridine was used as a starting material.

Example 4 Preparation of Sodium2,3,3-Trimethyl-1-(3-sulfonatopropyl)-3H-indolium-5-sulfonate (4)

Sodium 2,3,3-Trimethyl-1-(3-sulfonatopropyl)-3H-indolium-5-sulfonate 4)

A mixture of 14 g of sodium 2,3,3-trimethyl-3H-indole-5-sulfonate and 14g 1,3-propanesultone in 100 mL dicholorobenzene was heated at 110° C.for 2 h. After it cooled down, the solvent is decanted. The resultingsolid was then dissolved in 100 mL of acetonitrile, and 300 mL of ethylacetate was added. The resulting sticky solid was again stirred in 300mL of ethyl acetate to yield 20 g of the product.

Example 5 Preparation of3-(5-Chloro-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridin-7-ium-7-yl)propane-1-sulfonate(5)

3-(5-Chloro-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridin-7-ium-7-yl)propane-1-sulfonate(5)

A mixture of 1 g of 5-chloro-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridineand 1.60 g of 1,3-propanesultone was heated at 65° C. for 2 h. Ethylacetate (ca. 100 mL) was added and the resulting mixture was stirred atroom temperature overnight to yield 2.50 g of the product.

Example 6 Preparation of3-(5-Bromo-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridin-7-ium-7-yl)propane-1-sulfonate(6)

3-(5-Bromo-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridin-7-ium-7-yl)propane-1-sulfonate(6)

Compound 6 was prepared analogously to compound 5 (Example 5), exceptthat 5-bromo-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine (50) was used asa starting material.

Example 7 Preparation of3-(2,3,3-Trimethyl-3H-pyrrolo[2,3-b]pyridin-7-ium-7-yl)propane-1-sulfonate(7)

3-(2,3,3-Trimethyl-3H-pyrrolo[2,3-b]pyridin-7-ium-7-yl)propane-1-sulfonate(7)

A mixture of 9 g of 2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridine and 20.6 gof 1,3-propanesultone is heated at 60° C. for 3 h. The reaction mixtureis then dissolved in 100 mL of acetonitrile, and 300 mL of ethyl acetateis added. The resulting sticky solid is again stirred in 300 mL of ethylacetate to yield 22 g of the product.

Example 8 Preparation of(E)-N—((Z)-2-Bromo-3-(phenylamino)allylidene)benzenaminium Bromide (8)

(E)-N—((Z)-2-Bromo-3-(phenylamino)allylidene)benzenaminium Bromide (8)

The procedure as disclosed in the literature (Simonis, H. Ber. Deut.Chem. Ges. 1901, 34, 509; U.S. Pat. No. 6,747,159) is used. 3.54 g ofaniline are dissolved in 15 mL of ethanol in a 100 mL beaker.Separately, 5 g of mucobromic acid are dissolved in 15 mL of ethanol ina 100 mL Erlenmeyer flask. This solution is added dropwise to theaniline/ethanol solution, with cooling. The reaction mixture turnsimmediately yellow, then orange, with development of CO₂. At the end ofthe addition, the mixture is heated in a water bath until its volume isreduced by one half. The resulting solution is cooled with an ice-saltmixture, forming a yellow crystalline precipitate. This solid iscollected on a fritted glass filter to afford a first fraction of pureproduct 5 (3.84 g, 52% yield). Additional product 5 can be recoveredfrom concentration and recrystallization of the mother liquor.

Example 9 Preparation of Sodium(E)-2-((2Z,4E)-3-Bromo-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(9)

Sodium(E)-2-((2Z,4E)-3-Bromo-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(9)

A 100-mL round-bottom flask fitted with a reflux condenser was chargedwith3-(5-chloro-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridin-7-ium-7-yl)propane-1-sulfonate(55, 500 mg), sodium2,3,3-trimethyl-1-(3-sulfonatopropyl)-3H-indolium-5-sulfonate (4, 500mg), (E)-N—((Z)-2-bromo-3-(phenylamino)allylidene)benzenaminium bromide(8, 100 mg), pyridine (1 mL), and acetic anhydride (10 mL) were added tothe flask. The mixture was heated at 115° C. for 2 h, allowed to cool toroom temperature, and diluted with ethyl ether (25 mL). The resultingdark blue dye precipitate was collected by filtration, dissolved inwater (20 mL), and purified by preparative reverse-phase HPLC to affordthe 9 as a blue powder (285 mg, 50%, UV 668 nm).

Example 10 Preparation of Sodium(E)-2-(2Z,4E)-3-(3-(2-Carboxyethyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-1)]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(10)

Sodium(E)-2-(2Z,4E)-3-(3-(2-Carboxyethyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(10)

Compound 9 (80 mg), 3-(2-carboxyethyl)phenylboronic acid (40 mg), andcesium carbonate (20 mg) were stirred into water (20 mL) under nitrogenat room temperature. Tetrakis(triphenylphosphine)palladium (0) (10 mg)were added to the reaction mixture. The mixture was refluxed for 4 h,and the solvent and volatile compounds then were evaporated undervacuum. The crude product was purified by flash chromatography on silica60, 200-400 mesh, eluting with a 20/80 acetonitrile/water mixture. Thepurified compound 56 had λ_(MeOH)=680 nm, λ_(PBS)=672 nm, ε=160,000. Theabsorption and emission data is shown in Table 2 and FIG. 1.

TABLE 2 Absorption and Emission of Compound 10 Extinction Max. Abs. Max.Emis. Coefficient (nm) (nm) PBS 160,000 672 694 MeOH 170,000 680 694

Example 11 Preparation of Sodium(E)-2-((2Z,4E)-5-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-3-(3-(3-(2,5-dioxopyrrolidin-1-yloxy)-3-oxopropyl)phenyl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(11)

Sodium(E)-2-((2Z,4E)-5-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-3-(3-(3-(2,5-dioxopyrrolidin-1-yloxy)-3-oxopropyl)phenyl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(11)

To compound 10 (200 mg) and dry DMSO (15 mL) was added triethylamine(150 μL) and N,N′-disuccinimidyl carbonate (82 mg). The mixture wasstirred at room temperature for 2 h and then precipitated into diethylether (100 mL). The resulting solid was dried under vacuum to yield theN-hydroxy succinimidyl ester.

Example 12 Preparation of Sodium(E)-2-((2Z,4E)-3-(4-(2-Carboxyethyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropypindoline-5-sulfonate(12)

Sodium(E)-2-((2Z,4E)-3-(4-(2-Carboxyethyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(12)

Compound 12 is prepared analogously to compound 10 (Example 10), exceptthat 4-(2-carboxyethyl)phenylboronic acid is used as a startingmaterial.

Example 13 Preparation of Sodium(E)-2-((2Z,4E)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-3-(4-(3-(2,5-dioxopyrrolidin-1-yloxy)-3-oxopropyl)phenyl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(13)

Sodium(E)-2-((2Z,4E)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-3-(4-(3-(2,5-dioxopyrrolidin-1-yloxy)-3-oxopropyl)phenyl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(13)

Compound 13 is prepared analogously to compound 11 (Example 11), exceptthat compound 12 is used as a starting material.

Example 14 Preparation of Sodium(E)-2-((2Z,4E)-5-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-3-(3-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino)-3-oxopropyl)phenyl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(14)

Sodium(E)-2-((2Z,4E)-5-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-3-(3-(3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino)-3-oxopropyl)phenyl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(14)

The NHS ester dye 11 (0.18 mmol) is dissolved in 20 mL of dry DMSO andstirred at room temperature under dry nitrogen. Next, 2-maleimidio ethylamine (93.2 mg, 0.37 mmol) is added to the stirred solution, followed bydiisopropyl ethyl amine (DIPEA) (95 mg, 0.55 mmol). The stirring iscontinued for 45 min. DMF (20 mL) is added to the reaction, and stirringcontinued until thorough mixing is achieved. The solution is then pouredslowly into 400 mL of stirred diethyl ether to precipitate the product.The ether suspension is stirred for an additional 5 min and then allowedto stand for 1 hr. The ether is decanted, and an additional 20 mL of DMFis added to redissolve the solid. The DMF solution is then precipitatedinto a second 400 mL portion of stirred ether. The crude product iscollected by filtration. Optionally, further purification can beperformed, for example, by HPLC, column chromatography, orrecrystallization.

Example 15 Preparation of Sodium(E)-2-((2Z,4E)-3-(4-carboxyphenyl)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(15)

Sodium(E)-2-((2Z,4E)-3-(4-carboxyphenyl)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(15)

Compound 15 is prepared analogously to compound 10 (Example 10), exceptthat 4-carboxyphenylboronic acid is used as a starting material.λ_(MeOH)=680 nm.

Example 16 Preparation of Sodium2-((E)-2-((E)-2-Chloro-3-((E)-2-(3,3-dimethyl-7-(3-sulfonatopropyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indolium-5-sulfonate(16)

Sodium2-((E)-2-((E)-2-Chloro-3-((E)-2-(3,3-dimethyl-7-(3-sulfonatopropyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indolium-5-sulfonate(16)

Compound 16 is prepared analogously to compound 9 (Example 9), exceptthat compound 4, compound 7, andN-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]anilinemonohydrochloride are used as starting materials.

Example 17 Preparation of Sodium(E)-2-((E)-2-(2-(4-Carboxyphenyl)-3-((E)-2-(3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)vinyl)cyclohex-2-enylidene)ethylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(17)

Sodium(E)-2-((E)-2-(2-(4-Carboxyphenyl)-3-((E)-2-(3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)vinyl)cyclohex-2-enylidene)ethylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(17)

Compound 17 is prepared analogously to compound 10 (Example 10), exceptthat 4-carboxyphenylboronic acid and compound 16 are used as startingmaterials.

Example 18 Preparation of(E)-N-((E)-4-Carboxy-2-((phenylamino)methylene)butylidene)benzenaminiumchloride (18)

Compound 18 is made by using a modified procedure from W.I.P.O. PatentPublication WO 2010/054330s, which is incorporated by reference. (Seealso its priority document, U.S. Provisional Patent Application61/112,535 (filed Nov. 7, 2008) and Jauer, E. A.; Foerster, E.; Hirsch,B. Journal Fuer Signalaufzeichnungsmaterialien 1975 3(2) 155-163, bothof which are also incorporated by reference.) Anhydrousdimethylformamide (1.01 mL, 13 mmol) and phosphorus oxychloride (1.53 g,10 mmol) are added sequentially into 30 mL anhydrous dichloromethane(DCM) in an acetone/dry ice bath. The mixture is allowed to warm to roomtemperature over 15 min. Methyl 5,5-dimethoxyvalerate (881 mg, 5 mmol)is then added dropwise to the reaction solution followed by heating at70° C. for 2 h, allowing the DCM to evaporate. The resulting yellow oilis dissolved in 5 mL of 4 M aqueous NaOH, and the solution and is heatedat 70° C. for 1 hour. Next, with constant cooling at 20° C., anline/EtOH[1:1, (v/v), 10 mL] is added dropwise. The reaction is continued for anadditional 30 min after aniline addition, and then the yellow mixture ispoured into ice-cold water/concentrated aqueous HCl (10:1, 11 mL). Thefinal malonaldehyde dianil hydrochloride salts are precipitated as lightyellow solids after the addition of 5 mL of 10% aqueous HCl and werecollected by filtration. The products are above 95% pure and are useddirectly in the dye synthesis without further purification if notmentioned specifically.

Example 19 Preparation of Sodium(E)-2-((2E,4E)-3-(2-Carboxyethyl)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(19)

Sodium(E)-2-((2E,4E)-3-(2-Carboxyethyl)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(19)

Compound 19 is prepared analogously to compound 9 (Example 9), exceptthat compound 18 is used as a starting material. λ_(MeOH)=680 nm.

Example 20 Preparation of Compound 11-Streptavidin Conjugates

Compound 11 is reconstituted in DMF to 1 mg/mL. Streptavidin isreconstituted typically at 10 mg/ml in PBS buffer (pH 8.5). The dyes areadded (at various molar excesses) to the streptavidin samples andallowed to incubate for 2 h at room temperature in the dark. Theconjugates are extensively dialyzed against PBS buffer to remove theunconjugated free dye. The ratio of moles of dye per mole of protein iscalculated by using the equation below.

${D/P} = {\left\lbrack \frac{A_{685}}{ɛ_{Dye}} \right\rbrack \div \left\lbrack \frac{A_{280} - \left( {0.07 \times A_{685}} \right)}{ɛ_{Streptavidin}} \right\rbrack}$

In which:

ε_(dye)=160,000 M⁻¹cm⁻¹

ε_(streptavidin)=175,000 M⁻¹cm⁻¹

0.07 is the correction factor for the dye absorption at 280 nm

Example 21 Western Blot Comparison of GAM/IRDye® 20 and GAM/10Conjugates

Western blots were performed to compare GAM/IRDye 20 (GAM/20) and GAM/10(GAM/10) conjugates against a GAM/IRDye® 680 control (GAM/680). Theblots were probed with two different primary antibodies to determine ifthe extra bands were specific or were caused by dye sticking to thelysate.

Jurkat lysate was run (5 μg to 78 ng) by SDS PAGE and transferred tonitrocellulose. Blots were blocked with Odyssey Blocking Buffer+0.2%Tween 20 (OBBT). Blots were probed with either monoclonal anti-actin(Neomarkers MS-1295-P1) or monoclonal anti-tubulin (Sigma T7816) dilutedin OBBT. Blots were then detected with one of the following secondaryantibodies diluted in OBBT to a final concentration of 0.1 μg/ml:GAM/20, D/P=1.6 (Lot JE524089); GAM/10, D/P=1.6 (Lot JE524089); orGAM/680.

The blots are shown in FIG. 2. A summary of intensities are listed inTable 3 below:

TABLE 3 Western Blot Comparison of Total Fluorescence of GAM/20, GAM/10,and GAM/680 Antibody Bioconjugates Mean I.I. (K In- % % Bkgd Anti-Counts) Bkgd tensity Std Intensity of Blots tubulin Sample Sample SampleDev. of Control Control 1 GAM/ 82.73 233 74.21 12.06 control control 6802 65.68 243 3 GAM/ 171.16 262 191.85 29.26 259 108 20 4 212.54 250 5GAM/ 119.28 223 125.58 8.91 169  99 10 6 131.88 249 7 GAM/ 18.21 24617.24 1.37 control control 680 8 16.27 255 9 GAM/ 42.57 274 39.89 3.79231 110 20 10 37.21 275 11 GAM/ 18.51 255 21.73 4.55 126 100 10 12 24.95248The signal intensities are expressed in arbitrary fluorescence units(“Counts”), with the sample values divided by 1000 for convenience (“KCounts”) after subtraction of background. In general, protein detectionand quantitation are enhanced by increased fluorescence intensity and bylow fluorescence background.

It appears that the GAM/20 is about 2× more intense than the GAM/680control and the GAM/10 is about 1× more intense than the control. Bothsamples have comparable backgrounds to the control. The limit ofdetection was not determined.

Example 22 In-Cell Western Evaluation of GAM/20 and GAM/10

The low fluorescence background for biological materials in the NIRenables experiments in live or fixed cells in microplates. An importantexample is the In-Cell Western (“ICW”) technique, a cell-basedimmunohistochemical assay of cellular proteins in fixed cells. In suchsystems it is important that the dye-labeled antibody used for detectionmaintains the very low fluorescence background of the original cellularenvironment. Thus, the dye molecules attached to the detection antibodymust have very low non-specific binding to other cellular proteins, tomembranes, etc., or the labeled antibody will stick to those featuresand ruin the experiment.

The GAM bioconjugate with compounds 20 and 10 were evaluated by ICW.LI-COR IRDye® 680/GAM bioconjugate (i.e., LI-COR Part No. 926-32220) andAlexaFluor® 680 (GAM/AF-680) were used as controls. A431 cells, ATCCPart No. CRL-1555, were seeded in a 96 well plate and incubated at 37°C. for 48 hours. Cells were then fixed with 37% formaldehyde andpermeabilized with PBS+0.1% Triton® X-100. After permeabilization, cellswere blocked with Odyssey® Blocking Buffer.

GAM/compound 10 and GAM/compound 20 conjugates were both diluted inOdyssey® Blocking Buffer+0.2% Tween® 20 and added to the plate at afinal concentration of 2 μg/mL, 12 wells per sample. Samples wereincubated with gentle shaking at room temperature for 1 hour. The platewas washed three times with PBS+0.2% Tween® 20 and scanned on a LI-COROdyssey® Infrared Imager, using the Microplate 2 preset. The averageintegrated intensity was calculated for each sample and the compound 10-and 20-GAM antibody was compared to the controls. Average backgroundvalues were calculated and compared to controls. The results are listedin Tables 4 and 5 below.

TABLE 4 Comparison of Average Background Values for Example 25 SampleGAM/680 GAM/AF-680 GAM/20 GAM/10 OBB Only Average 4.07 4.35 5.24 4.024.08 Std. 0.21 0.61 1.37 0.12 0.14 Dev. % CV 5.06 14.00 26.19 2.89 3.51

TABLE 5 Comparison of Average Intensities for Example 25 Ave. <200%Sample Antibody Intensity % Control Control? 1 GAM/680 4.07 controlcontrol 2 GAM/20 5.24 129 yes 3 GAM/10 4.02 99 yes 4 GAM/AF-680 4.35control control 5 GAM/20 5.24 120 yes 6 GAM/10 4.02 92 yes

The inventive dyes (20/21, 10/11) performed well. The backgrounds andsignal intensities were comparable to those for the commercial antibodyconjugates.

Antibodies labeled with IRDye® 680 or with Alexa Fluor® 680 maintainvery low non-specific binding to cells. The data of Table 4 demonstratethat the GAM antibody labeled with compound 20 also has very lownon-specific background as good as or better than the other dyes tested.This dye can be used to produce labeled antibodies suitable for ICW andother cell-based (in vivo) applications.

Example 23 Preparation of sodium2-((2S,5R,8S,11S)-5-benzyl-8-(4-(3-(3-((1E,3Z,5Z)-1-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-5-(3,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)indolin-2-ylidene)penta-1,3-dien-3-yl)phenyl)propanamido)butyl)-11-(3-guanidinopropyl)-3,6,9,12,15-pentaoxo-1,4,7,10,13-pentaazacyclopentadecan-2-yl)acetate(21)

Sodium2-((2S,5R,8S,11S)-5-benzyl-8-(4-(3-(3-((1E,3Z,5Z)-1-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-5-(3,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)indolin-2-ylidene)penta-1,3-dien-3-yl)phenyl)propanamido)butyl)-11-(3-guanidinopropyl)-3,6,9,12,15-pentaoxo-1,4,7,10,13-pentaazacyclopentadecan-2-yl)acetate(21)

The NHS ester dye 13 (0.18 mmol) is dissolved in 20 mL of dry DMSO andstirred at room temperature under dry nitrogen. Next, the cyclicpentapeptide cyclo (Arg-Gly-Asp-D-Phe-Lys) (0.37 mmol) is added to thestirred solution, followed by diisopropyl ethyl amine (DIPEA) (95 mg,0.55 mmol). The stirring is continued for 45 min. DMF (20 mL) is addedto the reaction, and stirring continued until thorough mixing isachieved. The solution is then poured slowly into 400 mL of stirreddiethyl ether to precipitate the product. The ether suspension isstirred for an additional 5 min and then allowed to stand for 1 hr. Theether is decanted, and an additional 20 mL of DMF is added to redissolvethe solid. The DMF solution is then precipitated into a second 400 mLportion of stirred ether. The crude product is collected by filtration.Optionally, further purification can be performed, for example, by HPLC,column chromatography, or recrystallization.

Example 24 Evaluation of 10 for In Vivo Optical Imaging

The suitability of compound 10 for in vivo optical imaging was evaluatedusing an 10-labeled cyclo(Arg-Gly-Asp-D-Phe-Lys) (RGDfK) probe. Thisprobe was specifically designed to target integrins. Integrins are cellsurface heterodimeric glycoproteins important in cell adhesion andsignal transduction. This receptor class is involved in tumor growth,tumor invasiveness, metastasis, tumor-induced angiogenesis,inflammation, osteoporosis, and rheumatoid arthritis.

Mice were implanted with U87 tumor cells. The animals were injected andimaged while the tumors were still extremely small. This provided a goodtest of how visible the tumors are over any background signal that theprobe detected. FIG. 3 shows the image series (lateral view) of two miceafter receiving 1 nmol 10-labeled cyclo(Arg-Gly-Asp-D-Phe-Lys).

Tumors were excised, weighed, and imaged on a Pearl Imager (FIG. 3).These tumors were extremely small (9 and 15 mg), but were still easilydetectable by the procedure.

The agent cleared the system without an undue level of background whichwould obscure the tumors. These results demonstrate the suitability ofcompound 10 conjugates (made, e.g., by reaction with NHS ester 13 as inExample 23) for in vivo optical imaging.

Example 25 Compound 10 NHS Labeling and Western Blots

This method is generally applicable for the conjugation ofamino-containing molecules with NHS esters of inventive dye embodiments.As such, it provides a route to make the starting materials for manysubsequent examples such as the Western blot experiments withantibody/dye conjugates.

Goat anti-mouse antibody (Southern Biotech) was labeled withresearch-grade 12 NHS ester dye (i.e., 13). Compound 13 wasreconstituted in water to 1 mg/mL. Goat anti-mouse (GAM) IgG (H+L) werereconstituted typically at 1 mg/mL in phosphate buffer (pH 8.5). Thedyes were added (at various molar ratios, e.g., 2, 4, 6, 8, or 10) tothe GAM antibody samples and allowed to incubate for 2 hours at roomtemperature in the dark. If the reaction pH is too low, the amidecoupling reaction will be inefficient, and the dye to protein (D/P)ratios will be much lower than expected. If necessary, additionalequivalents of NHS ester can be used to drive the reaction tocompletion.

To purify the products, the free dye was removed by HPLC using PierceZeba columns. Alternatively, the conjugates are extensively dialyzedagainst phosphate buffered saline (1×PBS) to remove the unconjugatedfree dye.

Table 6 shows the absorbance results of all conjugates.

TABLE 6 Absorbance Readings and Calculation Results for Expanded Set of10 NHS Dye Conjugations with GAM. GAM conjugate Abs (280) Abs (680) D/PConc. (mg/mL)  2 equiv 0.12088 0.08880 0.95 0.87  4 equiv 0.143870.16139 1.47 1.03  6 equiv 0.14730 0.22814 2.06 1.04  8 equiv 0.152030.29371 2.60 1.06 10 equiv 0.1284 0.34010 3.65 0.87 10 + 5 equiv (10B)0.11354 0.35807 4.42 0.76

Three conjugates with a low, medium, and high D/P ratio (1.47, 2.60, and4.42 respectively) were tested in Western blots according to theprocedure set forth in Example 21. Compound 20, IRDye® 680, and AlexaFluor® 680 dye conjugates with GAM antibodies were included forcomparison as controls. The results are shown in FIGS. 5, 6A, and 6B.

Example 26 Imaging with 10/BoneTag™ Dye Conjugate (4 nmol, IV or IP)

A conjugate of 10 and BoneTag™ was prepared by reaction of the NHS esterof 10 with BoneTag™. The NHS ester of the dye was conjugated to atetracycline derivative containing a primary, aliphatic amine (pH 8, 25°C., 2 h). The product was purified by reverse-phase HPLC, aliquoted intotubes (20 nmol per tube), and lyophilized. As a skilled artisan willappreciate, binding of tetracycline and its derivatives to bone is wellknown in the art. For example, tetracycline, oxytetracycline,chlortetracycline and (1-pyrrolidinylmethyl)-tetracycline are taken upin newly-formed bone after injection into the living organism, to form azone that is intensely fluorescent under ultra-violet light. Thisreaction, which occurs wherever there is active deposition of new bone,and can also be used for the detection of calcification. See, Perrin,Nature 208, 787-788 (20 Nov. 1965).

For the animal studies, a portion of the conjugate was redissolved in1×PBS, and it was then filtered through a sterile, 0.1 micron filter.Nude mice were each injected with 4 nmol of conjugate and imaged 24 hlater with a Pearl® Impulse infrared imaging system. FIG. 7 shows theresults of intravenous (IV) and intraperitoneal (IP) administration.Bone structures are clearly visible in the resulting images because ofthe labeled tetracycline's binding to bone tissue.

Example 27 Lymph Imaging with 10/HA (2 nmol, ID)

A commercial sample of hyaluronan (approximately 30 kDal) was reactedwith hexamethylene diamine as described in U.S. Pat. No. 7,196,180 toproduce an aminohyaluronan derivative. This aminohyaluronan was reactedwith the NHS ester of 10 in aqueous solution (pH 8.5, 25° C., 3 h). Theproduct was purified with a spin column followed by dialysis, aliquotedinto tubes, and lyophilized.

For the animal studies, a portion of the conjugate was redissolved in1×PBS, and it was then filtered through a sterile, 0.1 micron filter.Three different applications for the agent were evaluated: i)intradermal, to watch lymph flow; ii) intravenous, to systemically labellymph nodes; and iii) intravenous, to target a tumor. The mice wereimaged in a Pearl Imager.

The sentinel nodes of the mice were highlighted in a Pearl Imager after10 min (FIG. 8) and after approximately 96 hours (FIG. 9). FIG. 8illustrates how an dose of the agent (˜0.5 nmol/3 μL) introducedintradermally at the base of the tail is picked up and pulsed to thenearest lymph nodes. FIG. 9 illustrates the systemic deposition of theagent in a number of lymph nodes. Panels A and B are non-invasive imagesof the subilliac and illiac/siatic lymph nodes. Panel C is four imagesof the same mouse after sacrifice and dissection to visualize deep nodesalong the aorta.

Example 28 Tumor Imaging with 10/HA (1 nmol, ID)

A conjugate of 10 and hyaluronan was prepared according to the procedureof Example 27. The conjugate (1 nmol) was intravenously administered toa nude mouse bearing a CD44-expressing tumor cell xenograft. The mousewas imaged in a Pearl Imager after approximately 24 h (FIG. 10).

Example 29 Localization of 10-PEG in A431 Tumor Xenograft

A commercial sample of mono-amino polyethylene glycol (mPEG, 40 kDal)was reacted with the NHS ester of 10 in aqueous solution (pH 8.5, 25°C., 2 h). The product was purified with a spin column, aliquoted andlyophilized.

For the animal studies, a portion of the conjugate was redissolved in1×PBS, and filtered through a sterile, 0.1 micron filter. Two nude micebearing A431 xenograft tumors were injected with the conjugate (1 nmol,IV) and imaged over time with a Pearl® Impulse infrared imaging system.The mice were imaged after approximately 1 min (FIG. 11) andapproximately 24 h (FIG. 12). The labeled mPEG accumulated in the tumordue to the malformed blood vessels there, allowing the tumor to bedetected at 24 h post-injection (FIG. 12).

One characteristic for the labeled PEG agents is the ability to seevasculature early after administration for approximately 30 minpost-injection. The vasculature around the tumor itself could beobserved. 10-PEG was effective as a tumor-imaging agent, and even highbackground did not overwhelm the tumor signal.

Example 30 Preparation of 6-Hydrazino-1,3-naphthalene Disulfonated Salt

6-Hydrazino-1,3-naphthalene Disulfonated Salt (22)

6-Amino-1,3-naphthalene disulfonate disodium salt (25 g, 72 mmol) wasdissolved in 150 mL of water and added to 50 mL of concentratedhydrochloric acid. The slurry was cooled to about 0° C. in an ice/saltbath, and sodium nitrite (5.46 g, 79.2 mmol) was added in 25 mL of coldwater dropwise over 10 minutes. Stannous chloride (20.42 g, 108 mmol)was dissolved in 15 mL concentrated hydrochloric acid, cooled to 0° C.and added to the reaction mixture over 20 minutes. The resultingsolution was allowed to warm to room temperature with stirring over 3hours. The solution was reduced in volume by rotary evaporation, and theproduct was precipitated by the addition of isopropanol. Compound 22 wasfiltered, washed with isopropanol, and dried under vacuum.

Example 31 Preparation of 2,3,3-Trimethylbenzindole-6,8-disulfonate Salt

2,3,3-Trimethylbenzindole-6,8-disulfonate Salt (23)

6-Hydrazino-1,3-naphthalene disulfonated salt 1 (10 g, 25 mmol),isopropyl methyl ketone (12 g, 140 mmol) and potassium acetate (6 g, 61mmol) were combined in 75 mL glacial acetic acid and heated to 145° C.for 22 hours. The solution was cooled, and the acetic acid was removedby rotary evaporation. The residue was dissolved in methanol andfiltered. The compound 23 was then precipitated from the methanolfiltrate with isopropanol, filtered, washed with isopropanol and ether,and dried under vacuum.

Example 32 Preparation of Sodium1,1,2-Trimethyl-3-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate

Sodium1,1,2-Trimethyl-3-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate(23)

2,3,3-Trimethylbenzindole-6,8-disulfonate 2 (2.2 g, 5 mmol) was stirredin 50 mL of dry 1,2-dichlorobenzene. 1,3-propanesultone (2.8 g, 23 mmol)was added, and the solution was heated to 145° C. in a sealed tube for15 hours. The solution was cooled, and the solvent was decanted off. Thesolid product 3 was washed on a filter with three 50 mL portions ofisopropanol followed by 50 mL of ether and dried under vacuum, resultingin a dark purple solid (2.5 g, 90%).

Example 33 Preparation of Sodium1,1,2-Trimethyl-3-(3-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate

Sodium1,1,2-Trimethyl-3-(3-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate(24)

Compound 24 was prepared analogously to compound 23 (Example 23), exceptthat 1,4-butanesultone is used as a starting material.

Example 34 Preparation of Sodium2-((1E,3Z,5E)-3-Bromo-5-(1,1-dimethyl-6,8-disulfonato-3-(3-sulfonatopropyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dienyl)-1,1-dimethyl-3-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate

Sodium2-((1E,3Z,5E)-3-Bromo-5-(1,1-dimethyl-6,8-disulfonato-3-(3-sulfonatopropyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dienyl)-1,1-dimethyl-3-(3-sulfonatopropyl)-1H-benzo[e]indolium-6,8-disulfonate(25)

Compound 25 is prepared analogously to compound 9 (Example 9), exceptthat compound 23 is used as a starting material.

Example 35 Preparation of4-(5-Chloro-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridin-7-ium-7-yl)butane-1-sulfonate

4-(5-Chloro-2,3,3-trimethyl-3H-pyrrolo[2,3-b]pyridin-7-ium-7-yl)butane-1-sulfonate(26)

Compound 26 is prepared analogously to compound 5 (Example 5), exceptthat 1,4-butanesultone is used as a starting material.

Example 36 Preparation of Sodium2-((1E,3Z,5E)-3-Bromo-5-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate

Sodium2-((1E,3Z,5E)-3-Bromo-5-(1,1-dimethyl-6,8-disulfonato-3-(3-sulfonatobutyl)-1H-benzo[e]indol-2(3H)-ylidene)penta-1,3-dienyl)-1,1-dimethyl-3-(3-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate(27)

Compound 27 is prepared analogously to compound 9 (Example 9), exceptthat compounds 24 and 26 is used as a starting material.

Example 37 Preparation of Sodium2-((1E,3Z,5E)-3-(3-(4-Carboxybutyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate

Sodium2-((1E,3Z,5E)-3-(3-(4-Carboxybutyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate(28)

Compound 27 (68 mg), 3-(4-carboxybutyl)phenylboronic acid (40 mg), andcesium carbonate (20 mg) are stirred into 1:1 water:ethanol (10 mL)under nitrogen at room temperature.Tetrakis(triphenylphosphine)palladium(0) (10 mg) is added to thereaction mixture. The mixture was refluxed for 4 hours, and the solventand volatile compounds are evaporated under vacuum. The crude product ispurified by flash chromatography on reverse-phase C18-functionalizedsilica by eluting with a 1:4 acetonitrile:water mixture.

Example 38 Preparation of Sodium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(5-(2,5-dioxopyrrolidin-1-yloxy)-5-oxopentyl)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate

Sodium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(5-(2,5-dioxopyrrolidin-1-yloxy)-5-oxopentyl)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate(29)

To a solution of compound 28 (200 mg) in dry DMSO (15 mL) is addedtriethylamine (150 μL) and N,N′-disuccinimidyl carbonate (82 mg). Themixture is stirred at room temperature for 2 hours, and the solvent isremoved to yield the succinimidyl ester.

Example 39 Preparation of Sodium2-((1E,3Z,5E)-3-(3-(2-Carboxyethyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate

Sodium2-((1E,3Z,5E)-3-(3-(2-Carboxyethyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate(30)

Compound 30 is prepared analogously to compound 28 (Example 37), exceptthat 3-(3-boronophenyl)propionic acid is used as a starting material.

Example 40 Preparation of Sodium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(3-(2,5-dioxopyrrolidin-1-yloxy)-3-oxopropyl)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate

Sodium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(3-(2,5-dioxopyrrolidin-1-yloxy)-3-oxopropyl)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate(31)

Compound 31 is prepared analogously to compound 29 (Example 38), exceptthat compound 30 is used as a starting material.

Example 41 Preparation of Sodium(E)-2-((2Z,4E)-3-(3-(4-Carboxybutyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate

Sodium(E)-2-((2Z,4E)-3-(3-(4-Carboxybutyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(32)

Compound 32 is prepared analogously to compound 10 (Example 10), exceptthat 3-(3-boronophenyl)butanoic acid is used as a starting material.

Example 42 Preparation of Sodium(E)-2-((2Z,4E)-5-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-3-(3-(5-(2,5-dioxopyrrolidin-1-yloxy)-5-oxopentyl)phenyl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate

Sodium(E)-2-((2Z,4E)-5-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-3-(3-(5-(2,5-dioxopyrrolidin-1-yloxy)-5-oxopentyl)phenyl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(33)

Compound 33 is prepared analogously to compound 29 (Example 38), exceptthat compound 32 is used as a starting material.

Example 43 Dot Blot Immunoassay Comparison of Total Fluorescence ofStreptavidin Bioconjugate of Compound 10 with Commercially AvailableStreptavidin Bioconjugates of IRDye® 680 and Alexa 680 Dye

Nitrocellulose membrane is spotted with different amounts ofbiotinylated anti-rabbit IgG. The membrane is blocked with LI-COROdyssey® Blocking Buffer for 30 min, followed by incubation for 30 minwith bioconjugates of compound 10 and streptavidin at different D/Pratios. The membrane is washed vigorously with 1×PBS and 1×PBS-T (1×PBSwith 0.1% Tween-20).

The membranes are scanned on a LI-COR Odyssey® Infrared Imager todetermine their fluorescence intensity (K Counts, with standardderivation), background intensity (including standard deviation), andlimit of detection.

Example 44 Western Blot Comparison of Total Fluorescence of StreptavidinConjugate to Compound 10 with Commercially Available Conjugates ofTRDye® 680 Dye to Streptavidin

Jurkat lysate is run on gels (5 μg to 78 ng). Blots are probed with msanti-actin (Thermo No. MS-1295P) diluted 1:1000 in Odyssey® BlockingBuffer+0.2% Tween® 20 followed by Biotin-SP GAM (Jackson No.115-065-166) diluted 1:20,000 in Odyssey® Blocker+0.2% Tween® 20. Blotsare then detected with various 680 streptavidin bioconjugates inOdyssey® Blocking Buffer+0.2% Tween® 20.

Example 45 Preparation of Sodium(E)-2-((2Z,4E)-5-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-3-(3-(5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino)-5-oxopentyl)phenyl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate

Sodium(E)-2-((2Z,4E)-5-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)-3-(3-(5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino)-5-oxopentyl)phenyl)penta-2,4-dienylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(34)

The NHS ester dye (33, 0.18 mmol) is dissolved in 20 mL of dry DMSO andstirred at room temperature under dry nitrogen. Next, 2-maleimidio ethylamine (93.2 mg, 0.37 mmol) is added to the stirred solution, followed bydi-isopropyl ethyl amine (DIPEA) (95 mg, 0.55 mmol). The stirring iscontinued for 45 min. DMF (20 mL) is added to the reaction, and stirringcontinued until thorough mixing is achieved. The solution is then pouredslowly into 400 mL of stirred diethyl ether to precipitate the product.The ether suspension is stirred for an additional 5 min, then allowed tostand for 1 hr. The ether is decanted and an additional 20 mL of DMF isadded to redissolve the solid. The DMF solution is then precipitatedinto a second 400 mL portion of stirred ether. The crude product iscollected by filtration. Optionally, further purification can beperformed, for example, by HPLC, column chromatography, orrecrystallization.

Example 46 Preparation of Sodium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino)-5-oxopentyl)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate

Sodium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(5-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino)-5-oxopentyl)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate(35)

Compound 35 is prepared analogously to compound 34 (Example 45), exceptthat compound 29 is used as a starting material.

Example 47 Preparation of Sodium(E)-2-((E)-3-((E)-2-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)vinyl)-6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohex-2-enylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate

Sodium(E)-2-((E)-3-((E)-2-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)vinyl)-6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohex-2-enylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(36)

Compound 36 is prepared analogously to compound 29 (Example 38), exceptthat compound 19 is used as a starting material.

Example 48 Preparation of Sodium(E)-2-((E)-3-((E)-2-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)vinyl)-6-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino)-6-oxohex-2-enylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate

Sodium(E)-2-((E)-3-((E)-2-(5-Chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)vinyl)-6-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethylamino)-6-oxohex-2-enylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(37)

Compound 37 is prepared analogously to compound 34 (Example 45), exceptthat compound 36 is used as a starting material.

Example 49 Preparation of Sodium2,3,3-Trimethyl-1-(3-sulfonatobutyl)-3H-indolium-5-sulfonate

Sodium 2,3,3-Trimethyl-1-(3-sulfonatobutyl)-3H-indolium-5-sulfonate (38)

Compound 38 is prepared analogously to compound 4 (Example 4), exceptthat 1,4-butanesultone is used as a starting material.

Example 50 Preparation of Sodium2-((E)-2-((E)-2-chloro-3-((E)-2-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)ethylidene)cyclopent-1-enyl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate

Sodium2-((E)-2-((E)-2-chloro-3-((E)-2-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)ethylidene)cyclopent-1-enyl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate(39)

Compound 39 is prepared analogously to compound 16, except with compound38, compound 26, and the cyclopentyl chloro dye precursor as startingmaterials.

Example 51 Preparation of Sodium2-((E)-2-((E)-2-(3-carboxyphenyl)-3-((E)-2-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)ethylidene)cyclopent-1-enyl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate

Sodium2-((E)-2-((E)-2-(3-carboxyphenyl)-3-((E)-2-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)ethylidene)cyclopent-1-enyl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate (40)

Compound 40 is prepared analogously to compound 17 (Example 17), exceptwith compound 39 and 3-boronobenzoic acid as starting materials.

The skilled person will appreciate that the boronic acid intermediatesused here are versatile and can be modified by custom synthesis to meetvarious design changes. The phenyl ring can be substituted with varioustypes of substituents and substituent lengths. One custom synthesismanufacture is Combi-Blocks, Inc. of San Diego, Calif.

Example 52 Preparation of Sodium2-((E)-2-((E)-2-(3-(3-carboxypropyl)phenyl)-3-((E)-2-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)ethylidene)cyclopent-1-enyl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate

sodium2-((E)-2-((E)-2-(3-(3-carboxypropyl)phenyl)-3-((E)-2-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)ethylidene)cyclopent-1-enyl)vinyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate(41)

Compound 41 is prepared analogously to compound 40 (Example 51), exceptwith 4-(3-boronophenyl)butanoic acid as a starting material.

Example 53 Preparation of Sodium2-((1E,3Z,5E,7E)-4-chloro-7-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)hepta-1,3,5-trienyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate

sodium2-((1E,3Z,5E,7E)-4-chloro-7-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)hepta-1,3,5-trienyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate(42)

Compound 42 is prepared analogously to compound 39 (Example 51), exceptwith a non-cyclopentyl chloro precursor as a starting material.

Example 54 Preparation of Sodium2-((1E,3Z,5E,7E)-4-(3-carboxyphenyl)-7-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)hepta-1,3,5-trienyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate

Sodium2-((1E,3Z,5E,7E)-4-(3-carboxyphenyl)-7-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)hepta-1,3,5-trienyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate(43)

Compound 43 is prepared analogously to compound 17 (Example 17), exceptwith compound 42 and m-carboxyphenyl boronic acid as starting materials.

Example 55 Preparation of Sodium2-((1E,3Z,5E,7E)-4-(3-(3-carboxypropyl)phenyl)-7-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)hepta-1,3,5-trienyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate

Sodium2-((1E,3Z,5E,7E)-4-(3-(3-carboxypropyl)phenyl)-7-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)hepta-1,3,5-trienyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate(44)

Compound 44 is prepared analogously to compound 43, except with4-(3-boronophenyl)butanoic acid as a starting material.

Example 56 Preparation of Sodium(E)-2-((E)-2-(3-((E)-2-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)vinyl)-2-(3-(4-hydroxybutoxy)phenyl)cyclohex-2-enylidene)ethylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate

Sodium(E)-2-((E)-2-(3-((E)-2-(5-chloro-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-2-yl)vinyl)-2-(3-(4-hydroxybutoxy)phenyl)cyclohex-2-enylidene)ethylidene)-3,3-dimethyl-1-(3-sulfonatopropyl)indoline-5-sulfonate(45)

Compound 45 is prepared analogously to compound 17, except with3-(4-hydroxybutoxy)phenylboronic acid as shown above.

Example 57 Preparation of Tetrabutylammonium2-((E)-2-((E)-3-((E)-2-(3,3-Dimethyl-5-sulfonato-1-(3-sulfonatopropyl)indolin-2-ylidene)ethylidene)-2-(3-(4-hydroxybutoxy)phenyl)cyclohex-1-enyl)vinyl)-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-5-sulfonate

Tetrabutylammonium2-((E)-2-((E)-3-((E)-2-(3,3-Dimethyl-5-sulfonato-1-(3-sulfonatopropyl)indolin-2-ylidene)ethylidene)-2-(3-(4-hydroxybutoxy)phenyl)cyclohex-1-enyl)vinyl)-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-5-sulfonate(46)

Compound 46 is prepared by ion exchange of the sodium ions of compound45. Ion exchange to salts such as tetralkylammonium and the like willimprove the solubility of the dye in organic solvents suitable for DNAsynthesis (e.g., acetonitrile). This can be done with chromatography aspart of the purification process for the hydroxy dye, or as a separateion-exchange step. Commercial cationic ion exchange resins are widelyavailable.

Example 58 Preparation of Tetrabutylammonium2-((E)-2-((E)-2-(3-(4-((2-Cyanoethyl)(diisopropylamino)phosphinooxy)butoxy)phenyl)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)indolin-2-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-5-sulfonate

Tetrabutylammonium2-((E)-2-((E)-2-(3-(4-((2-cyanoethoxy)(diisopropylamino)phosphinooxy)butoxy)phenyl)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(3-sulfonatopropyl)indolin-2-ylidene)ethylidene)cyclohex-1-enyl)vinyl)-3,3-dimethyl-7-(3-sulfonatopropyl)-3H-pyrrolo[2,3-b]pyridin-7-ium-5-sulfonate

The general procedure described in U.S. Pat. No. 6,027,709 is used.Compound 46 (0.140 mmol) is dissolved in 10 mL of dry methylene chlorideand stirred under argon at about 0° C. in an ice/salt bath for 30minutes. A solution of bis(N,N-diisopropylamino)-cyanoethyl phosphine(2.13 mL., 0.15 M in methylene chloride) is added to the dye solution.Tetrazole (0.128 mL., 0.5 M) in acetonitrile is then added to the cooledsolution. The cooling is removed after 20 minutes and the reaction iscontinued for an additional 1.5 hours at room temperature. The reactionmixture is quenched with 5% aqueous sodium bicarbonate solution, washedtwice with water, and dried with sodium sulfate. The solvent is removedunder vacuum. The crude product is taken up in 1.5 mL of methylenechloride, and the product 47 is obtained by precipitation into hexane.

Example 59 Preparation of Oligonucleotide Bioconjugate withPhosphoramide Linking Group

Oligonucleotide Bioconjugate with Phosphoramide Linking Group (48)

The general procedure described in U.S. Pat. No. 6,027,709 is used. Thephosphoramidite of the fluorescent dye 47 can be used to label DNAmolecules prepared in a DNA synthesis machine. The dye is attached tothe 5′ end of the protected, support-bonded oligonucleotide via standardphosphoramidite chemistry. Typical yields on a 200 nmol scale isexpected to range from 50 to 100 nmol before purification.

Each of the DNA oligonucleotides M13 fwd (−29), M13 rev, T7, T3 and SP6,is synthesized in the PerSeptive Biosystems Expedite 8909 DNA synthesismachine in accordance with standard reagents and the methodology taughtby the manufacturer. The same apparatus then is used to attach thefluorescent label to the 5′ end of each oligonucleotide by treatmentwith a 0.1 M solution of the dye phosphoramidite produced above inacetonitrile. For the attachment of the dye phosphoramidite, athree-minute delay is inserted after the delivery of the dye in thetetrazole to the synthesis column to allow additional time for thecoupling reaction. The 5′-fluorescent labeled DNA oligonucleotide isproduced following oxidation, cleavage, deprotection and purification byHPLC.

For HPLC purification of the labeled oligonucleotide, a C18reverse-phase column having 5μ particles, 300 A pore size (WatersDeltaPak), 1.7 mL/min may be used. Solvent A is 4% acetonitrile inaqueous 0.1 M triethylammonium acetate, and Solvent B is an 80%acetonitrile in aqueous 0.1 M triethylammonium acetate. The gradientprofile is 10 to 45% B over 35 minutes, 45 to 100% B over 15 minutes,100 to 10% B in 10 minutes. One of skill in the art may modify orreplace these conditions as necessary for purification of various dyes.

The labeled oligonucleotide bioconjugate 48 can be used, for example, asa primer in the Sanger method of DNA sequencing, as a tailed primer forgenotyping, or as a hybridization probe.

Example 60 Preparation of Sodium2-((1E,3Z,5E)-3-(3-(4-Carboxybutyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)penta-1,3-dienyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate

Sodium2-((1E,3Z,5E)-3-(3-(4-Carboxybutyl)phenyl)-5-(5-chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)penta-1,3-dienyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate(49)

Compound 49 is prepared analogously to compound 43 (Example 54), exceptwith 5-(2-boronophenyl)pentanoic acid as a starting material.

Example 61 Preparation of Sodium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(5-(2,5-dioxopyrrolidin-1-yloxy)-5-oxopentyl)phenyl)penta-1,3-dienyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate

Sodium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(5-(2,5-dioxopyrrolidin-1-yloxy)-5-oxopentyl)phenyl)penta-1,3-dienyl)-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indolium-5-sulfonate(50)

Compound 50 is prepared analogously to compound 29 (Example 38), exceptthat compound 49 is used as a starting material.

Example 62 Preparation of Sodium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(4-hydroxybutoxy)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate

Sodium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(4-hydroxybutoxy)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate(51)

Compound 51 is prepared analogously to compound 28, except with3-(4-hydroxybutoxy)phenylboronic acid as a starting material.

Example 63 Preparation of Tetrabutylammonium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(4-hydroxybutoxy)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate

Tetrabutylammonium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(4-hydroxybutoxy)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate(52)

Compound 52 is prepared analogously to compound 46 (Example 57).

Example 64 Preparation of Tetrabutylammonium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(4-((2-cyanoethyl)(diisopropylamino)phosphinooxy)butoxy)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate

Tetrabutylammonium2-((1E,3Z,5E)-5-(5-Chloro-3,3-dimethyl-7-(4-sulfonatobutyl)-3,7-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ylidene)-3-(3-(4-((2-cyanoethyl)(diisopropylamino)phosphinooxy)butoxy)phenyl)penta-1,3-dienyl)-1,1-dimethyl-3-(4-sulfonatobutyl)-1H-benzo[e]indolium-6,8-disulfonate(53)

Compound 53 is prepared analogously to compound 47 (Example 58).

Example 65 Preparation of Oligonucleotide Bioconjugate II withPhosphoramide Linking Group

Oligonucleotide Bioconjugate II with Phosphoramide Linking Group (54)

Compound 54 is prepared and used analogously to compound 48 (Example59).

Example 66 Preparation of Oligonucleotide Bioconjugate III withPhosphoramide Linking Group

Oligonucleotide Bioconjugate III with Phosphoramide Linking Group (55)

Compound 55 and its precursors are prepared analogously to compounds 9,10, and 45-48.

Example 67 Dye Brightness Compound 20

The baseline fluorescence of 20 was determined.

Fluorescence determinations were made at a fixed antibody concentrationof 10 μg/mL in physiological buffer using dye-labeled goat anti-rabbit(GAR) conjugates prepared at LI-COR (FIGS. 13A-B and 14). A dye-labeledlactalbumin conjugate was also tested (FIG. 15).

20's absorbance and emission maximum is at 676 nm in aqueous solutionand at 693 nm in methanol. 20 is a highly water-soluble dye optimizedfor use on the Odyssey Infrared Imager and the Aerius Automated Imagerin the 700 nm channel.

Example 68 Dye Brightness Compound 10

The baseline fluorescence of 10 is determined analogously to 20 (Example48).

Example 69 Comparison of Dye Brightness Compound 20 and AlexaFluor® 680

A comparison of 20 with the commercially available dye AlexaFluor® 680was conducted.

Fluorescence determinations were made at a fixed antibody concentrationof 10 mg/mL in physiological buffer using dye labeled goat anti-rabbit(GAR) conjugates prepared at LI-COR. Fluorescence was measured using aPTI Fluorometer at the optimum excitation and emission wavelength ofeach dye.

Fluorescence intensity was also tested with an Odyssey Infrared Imager(FIGS. 16 and 17). The fluorescence intensity of each conjugateincreased with increased degree of labeling. A plot of degree oflabeling versus fluorescence shows that the dynamic range for the 20/GARwas much broader than the AlexaFluor®, which quickly leveled off aboveD/P 3.4 (FIG. 18). The leveling off of the AlexaFluor® 680 conjugatesmay be due to self-quenching. Compound 20 conjugates continue toincrease in fluorescence intensity to at least D/P 6.4. Overall, 20 issignificantly brighter than AlexaFluor® 680.

Example 70 Comparison of Dye Brightness Compound 10 and AlexaFluor® 680

A comparison of dye brightness for 10 is determined analogously to 20(Example 69).

Example 71 Comparison of Photostability Compound 20, IRDye 700Dx, andAlexaFluor® 680

The photostability of 20 was compared to AlexaFluor®680 and IRDye 700Dx,which is considered to be one of the most stable 700 nm fluorescentdyes. Test samples were prepared by spotting equimolar amounts of dye(i.e., goat anti-rabbit (GAR) secondary antibodies labeled with theappropriate dye) onto nitrocellulose membrane. The membrane was thenscanned repeatedly on an Odyssey Infrared Imager, and the signalintensity was normalized to the control signal at time zero (FIG.19A-B).

The relative fluorescence of the 700DX samples was unchanged, the 680 LTfluorescence decreased slightly, and the AlexaFluor® 680 fluorescencedecreased significantly. Therefore, the most stable dye was 700DXfollowed by 20 and AlexaFluor® 680.

Example 72 Comparison of Photostability Compound 10 and AlexaFluor® 680

A comparison of photostability for 10 is determined analogously to 20(Example 71).

Example 73 Comparison of GAR Cell Staining Compound 20 and AlexaFluor®680

GAR secondary antibodies labeled with 20 or AlexaFluor® 680 forfluorescence measurements were used for cell staining (as previouslydescribed for the other GAR functional testing) (FIG. 20A-B).

Cultured SK-BR-3 (A) or SK-OV03 (B) were fixed with 3.7% formaldehydeand permeabilized with 0.1% Triton X-100. Cells were incubated withrabbit anti-HER2 mAb (CST), followed by goat and rabbit secondaryantibodies labeled with IRDy3 680 LT or Alexa Fluor 680. The images werescanned on an Odyssesy scanner. The original images are shown on theleft, and the quantified signal intensities are shown on the right (FIG.20A-D).

The brightness and photostability of 20 make it an excellent choice formicroscopy and In-Cell Westerns™. The dynamic range for 20 was widerthan for AlexaFluor® 680. As well, overall fluorescence intensity or“brightness” at comparable D/P ratios was greater for 20 than forAlexaFluor® 680. The signal intensity was two- to three-fold higher forcells stained with 20 labeled secondary antibody compared to theAlexaFluor® 680 conjugates. Later In-Cell Western data mimicked thefluorescence measurement data for the dye labeled conjugates.

Example 74 Comparison of GAR Cell Staining Compound 10 and AlexaFluor®680

A comparison of GAR cell staining for 10 is determined analogously to 20(Example 73).

Example 75 Comparison of Immunofluorescence Staining Compound 20 andAlexaFluor® 680

HER2 protein was stained with dye-labeled antibodies on SK-BR-3 cellmembrance. GAR secondary antibodies labeled with 20 were used forfluorescence measurements.

The cells were cultured on cover slips. After fixation andpermabilization as per Example 52, the cells were incubated with rabbitanti-HER2 mAb (CST), followed by 20-labeled GAR secondary antibody(D/P=3.3). Sytox green was used to stain the nuclei. Images wereacquired on an Olympus microscope and deconvolved using the accompanyingsoftware (FIG. 21A-C).

Example 76 Comparison of Immunofluorescence Staining Compound 10 andAlexaFluor® 680

A comparison of immunofluorescence staining for 10 is determinedanalogously to 20 (Example 75).

Example 77 Comparison of 13-Actin Western Blots Compound 20, IRDye® 680,and AlexaFluor® 680

Compound 20 conjugates were compared by Western blot to commerciallyavailable IRDye 680 and AlexaFluor® 680 goat anti-mouse (GAM) antibodyconjugates. AlexaFluor® 680 antibody conjugates are supplied at 2 mg/mLand were diluted to 0.1 mg/mL (1:20,000). The IRDye 78 secondaryantibodies are reconstituted to give a stock concentration of 1 mg/mLand were diluted to 0.1 mg/mL (1:10,000). A 1:20,000 dilution ofAlexaFluor® 680 GAM has approximately equivalent amount of protein as a1:10,000 dilution of the IRDye antibodies.

Western blots were performed to detect actin in C32 lysates. Two-folddilutions starting at 10 μg of C32 whole cell lysate (Santa Cruz) wereloaded on a 10% Bis-Tris reducing gel and transferred to anitrocellulose membrane (Odyssey). The mouse primary antibody againstβ-actin (Thermo Fisher Scientific) was used at 1:1000 dilution in abuffer of 0.2% Tween 20 in Odyssey Blocking Buffer. The various goatanti-mouse secondary antibodies were diluted in the same way. Allantibody incubation was for 1 hour at ambient temperature. The resultsof the Western blot are shown in FIG. 22A-C. All Western blots wereperformed in duplicate.

An assessment of signal intensity, background and working dilution ofthe 20 antibodies was made in comparison to both IRDye® 680 andAlexaFluor® secondary antibodies. (FIG. 13) Overall, the signal detectedwith 20/GAM is 3× greater than IRDye 680 and 1.5× higher thanAlexaFluor® 680 GAM. The background on membrances treated with 20/GAMwas comparable to that with the other 700 channel fluorophores. Visualinspection of the Western blots indicated a similar limit of detectionbetween the three GAM-conjugated antibodies. Also, the 20/GAM, diluted1:25,000, maintains superior performance in terms of signal whencompared to IRDye® 680 GAM at the same dilution.

Example 78 Comparison of β-Actin Western Blots Compound 10, IRDye® 680,and AlexaFluor® 680

A comparison of β-actin Western blots for 10 is determined analogouslyto 20 (Example 77).

Example 79 Comparison of p38 Western Blots Compound 20, IRDye® 680, andAlexaFluor® 680

A Western blot was performed to detect the lower expressing protein p38in Jurkat lysates. Conjugates of compound 20 were compared withcommercially available IRDye® 680 and AlexaFluor® 680 goat anti-rabbit(GAR) antibody conjugates. The dye-labeled antibodies were diluted asdescribed in Example 77.

Two-fold dilutions starting at 10 μg of Jurkat cell lysate were loadedon a 10% Bis-Tris reducing gel and transferred to a nitrocellulosemembrane (Odyssey). The rabbit primary antibody against p38 (Santa Cruz)was used at 1:1000 dilution in a buffer of 0.2% Tween 20 in OdysseyBlocking Buffer. The various goat anti-rabbit secondary antibodies werediluted in the same way. All antibody incubations were for 1 hour atambient temperature. The results of the Western blot are shown in FIG.24A-C. All Western blots were performed in duplicate.

The 20/GAR conjugates outperformed IRDye® 680 GAR with a 2× to 4×improvement in signal intensity. With this target, the visual limit ofdetection was also improved by the same factor (1:10,000-1:25,000dilutions, respectively). The signal of IRDye 680 GAR is 1.7-0.9× asbright as the AlexaFluor® 680 GAR (AlexaFluor® 680 1:20,000 dilution),and the limit of detect was within a single two-fold dilution for thep38 target. Additional bands were seen on all Western blots, indicatingthat the primary antibody is detecting additional proteins.

Example 80 Comparison of p38 Western Blots Compound 10, IRDye® 680, andAlexaFluor® 680

A comparison of p38 Western blots for 10 is determined analogously to 20(Example 79).

Example 81 Akt Two-Color Western Blot Compound 20, IRDye® 800CW, andAlexaFluor® 680

Balanced two-color Western blots to detect the low abundance protein Aktwere performed with 20/GAM and IRDye® 800CW GAR antibodies. Thedye-labeled antibodies were diluted as described in Example 58.

NIH 3T3 cell lysates (two-fold dilutions starting at 10 μg) wereseparated by SDS-PAGE and transferred to nitrocellulose. The membraneswere blocked with LI-COR Blocking Buffer. The primary antibodies wereagainst Akt (mouse mAb) and actin (rabit mAb), diluted 1:1000. Thesecondary antibody to detect actin was IRDye® 800CW GAR (1:10,000). Aktwas detected with (A) IRDye® 680 GAM (1:10,000); (B) AlexaFluor® 680 GAM(1:20,000); (C) 20/GAM 1:10,000) on Odyssey (700 channel=5; 800channel=5).

Once again, 20 conjugates were brighter compared to IRDye® 680 (FIG. 25Aand FIG. 25C). 20/GAM showed lower background than AlexaFluor® 680 GAMwith an equivalent visual limit of detection (FIG. 25B-C).

Example 82 Comparison of Akt Two-Color Western Blots Compound 10, IRDye®680, and AlexaFluor® 680

A comparison of Akt two-color Western blots for 10 is determinedanalogously to 20 (Example 81).

Example 83 Akt Western Blot with Compound 20

Additional Western blot experiments (FIG. 26A-B) were performed using20/GAM to detect Akt in A431 lysates. The dye-labeled antibodies werediluted as described in Example 77.

A431 lysates were separated on a 10% Bis-Tris gel, transferred toOdyssey nitrocellulose and blocked in Odyssey® Blocking Buffer. Forexperiment A, the membrane was incubated for 1 hour with Akt mAb (CellSignaling Technologies) diluted in Odyssey® Blocking Buffer (1:1000),washed, and then incubated for 1 hour with 20/GAM (1:10,000). It wasdiluted in Odyssey® Blocking Buffer including 0.2% Tween 20. Forexperiment FIG. 26B, the membrane was incubated only in secondaryantibody as described in A. All membranes were washed as directed andimaged on the Odyssey Infrared Imager.

FIG. 26A illustrates the linearity of the conjugate over a large rangeof protein concentrations (50 μg-20 ng; R² is 0.9982 from 30 μg to 20 nglysate).

Compound 20 secondary antibodies have been shown to have lownon-specific binding to proteins in a variety of lysates (Jurkat, HeLa,C32, A431 & NIH3T3). An example of this low binding is shown in FIG.26B, as the Western blot was performed without primary antibody. Even inthe presence of 50 μg of protein there is little signal detected fromthe 20/GAM antibody.

Example 84 Akt Two-Color Western Blot with Compound 10

An Akt Western blots for 10 is determined analogously to 20 (Example83).

Example 85

Example 85 illustrates the synthesis of2-((E)-2-((E)-3-((E)-2-(1-(1-azido-13-oxo-3,6,9-trioxa-12-azaoctadecan-18-yl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)ethylidene)-2-(4-sulfophenoxy)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium-5-sulfonate(IRDye 800CW-PEG-Azide, 56).

A solution of 11-azido-3,6,9-trioxaundecan-1-amine (Amino-PEG-Azide, 1.0mg, 4.1×10⁻³ mmol) and N,N-diisopropylethylamine (0.0020 mL, 1.1×10⁻²mmol) in anhydrous dimethyl sulfoxide (0.2 mL) was added to a reactionvessel containing IRDye 800CW NHS ester (5.0 mg, 4.3×10⁻³ mmol). Thereaction was allowed to proceed at ambient temperature for 2 hours, withperiodic agitation every 15 minutes. After HPLC analysis indicatedcomplete consumption of the IRDye 800CW NHS ester, the reaction mixturewas precipitated into anhydrous diethyl ether. The ethereal layer wasdecanted and the crude product was purified by reverse-phase HPLC.Fractions containing the desired IRDye 800CW-PEG-Azide in ≧95% purity byHPLC analysis were combined and lyophilized to afford the product 56 asa green flocculent solid (3.4 mg, 66% based on Amino-PEG-Azide). UV/Vis(methanol) λ_(max)=778 nm; LRMS (ES/water), m/z calculated for 1203.37[M+H]⁺. found 1203.6 and 602.3 [M+2H]²⁺.

Example 86

Example 86 illustrates a trityl-protected version of Compound 10(Compound 10-NH-(PEG)₂-NH-Trt, 57).

A solution ofO—(N-trityl-3-aminopropyl)-O′-(3-aminpropyl)-diethyleneglycol(Trt-NH-PEG₂-NH₂, 1.0 mg, 2.2×10⁻³ mmol) and N,N-diisopropylethylamine(0.001 mL, 5.7×10⁻³ mmol) in anhydrous dimethyl sulfoxide (0.2 mL) wasadded to a reaction vessel containing Compound 11 (1.0 mg, 1.0×10⁻³mmol). The reaction was allowed to proceed at ambient temperature for 2hours, with periodic vortexing at 15-minute intervals. After HPLCanalysis showed complete consumption of Compound 11, the reaction wasprecipitated into anhydrous diethyl ether. The ethereal layer wasdecanted and the crude product was purified by HPLC. Fractionscontaining the presumed Compound 10-NH-PEG₂-NH-Trt in ≧95% purity werecombined and concentrated in vacuo to afford a blue film; the yield waspresumed to be quantitative.

Example 87

Example 87 illustrates another derivative of Compound 10 (Compound10-NH-(PEG)₂-NH₂.2TFA, 58).

To a flask containing Compound 10-NH-PEG₂-NH-Trt (1.3 mg, 8.6×10⁻⁴) wasadded a solution of trifluoroacetic acid in dichloromethane(TFA/CH₂Cl₂=1:3, 5.0 mL). The purple reaction was briefly swirled andallowed to proceed at ambient temperature for 30 minutes. The volatileswere removed in vacuo and the residuals were treated again withTFA/CH₂Cl₂ (1:3, 5.0 mL) for 30 minutes. After removing the volatiles invacuo, the residuals were washed with anhydrous diethyl ether. Theethereal layer was decanted, and the product 58 was used without furtherpurification; the yield was presumed to be quantitative. UV/Vis(methanol) λ_(max)=676 nm; LRMS (water) m/z calculated for 1064.4[M+H]⁺. found 1064.6, 532.9 [M+2H]²⁺.

Example 88

Example 88 illustrates the synthesis of a phosphine Compound 10derivative (Compound 10-PEG-Phosphine, 59).

To a solution of Compound 10-NH-(PEG)₂-NH₂.2TFA (1.3 mg, 1.0×10⁻³ mmol)in anhydrous dimethyl sulfoxide (0.2 mL) was added NHS-Phosphine (1.0mg, 2.2×10⁻³ mmol, ThermoScientific/Pierce) followed byN,N-diisopropylethylamine (0.001 mL, 5.7×10⁻³ mmol). The reaction wasallowed to proceed at ambient temperature for 2 hours, with periodicagitation at 15-minute intervals. After HPLC analysis showednear-complete consumption of the Compound 10-NH-(PEG)₂-NH₂.2TFA, thereaction was precipitated into anhydrous diethyl ether. The ethereallayer was decanted and the crude product was purified by HPLC. Fractionscontaining the presumed Compound 10-PEG-Phosphine in ≧95% purity werecombined and concentrated in vacuo to afford a blue solid (0.7 mg, 51%based on Compound 10-NH-(PEG)₂-NH₂.2TFA); UV-Vis (methanol) λ_(max)=676nm; LRMS (water) m/z calculated for 1410.4 [M+H]% found 705.8 [M+2H]²⁺.

Example 89

Example 89 illustrates a phosphine oxide Compound 10 derivative(Compound 10-PEG-Phosphine Oxide, 60).

This compound was isolated as a substantial byproduct from the synthesisof Compound 10-PEG-Phosphine. This byproduct is nonfunctional and causesbackground problems. The compound is a blue solid (0.2 mg, 18% basedCompound 10-NH-(PEG)₂-NH₂.2TFA); UV-Vis (methanol) λ_(max)=676 nm; LRMS(water) m/z calculated for 1426.4 [M+H]⁺. found 1426.5, 713.9 [M+2H]²⁺.

Example 90

Example 90 illustrates the synthesis of a two-dye Staudinger ligationproduct (IRDye 800CW-Compound 10 Click Product, 61).

To a solution of IRDye 800CW-PEG-Azide (56) (55 μg, 4.3×10⁻² μmol) inwater (20 μL) was added a solution of Compound 10-PEG-Phosphine (59) (73μg, 5.0×10⁻² mmol) in water (150 μL). The reaction was allowed toproceed at ambient temperature for 1 hour, then maintained at 40° C. for3 hours. After HPLC analysis showed near-complete consumption of bothreagents, the reaction mixture was filtered and directly purified byHPLC. Fractions containing the presumed IRDye 800CW-Compound 10 ClickProduct were combined and concentrated in vacuo. The exact yield was notdetermined. UV/Vis (acetonitrile/water=1:1) λ_(max)1=778 nm,λ_(max)2=677 nm; LRMS (ES/water), m/z calculated for 2570.8 [M+H]⁺.found 858.5 [M+3H]³⁺.

Example 91

Example 91 illustrates a non-catalyzed click chemistry synthesisreaction.

Bertozzi et al. (Aldrichimica Acta, 2010, 43(i), 15-23 and referencestherein) have developed a bio-orthogonal labeling method that employsmodified sugars. Cells incubated in a growth medium containing thesemodified azido sugars will absorb the sugars and perhaps incorporate thesugars on cell surface glycans (i.e., glyco-proteins and glycolipids).Upon exposing the azido-sugar labeled cells to appropriate phosphine oralkyne reagents, a click-type reactions will occur (either a Staudingerligation or Huisgen cycloaddition, respectively). If the phosphine oralkyne bears a reporter group (e.g., a dye), then the cells are labeledas shown below in Scheme 1:

In this example, the reaction is performed with IRDye 800CW as the dye.The conjugate has the following advantages: (1) This type ofbio-orthogonal labeling does not entail genetic engineering. Althoughmany biological researchers study transgenic organisms, developmentalbiologists simply want to monitor “normal” changes in biochemicalmorphology. (2) The click reagents are highly chemoselective andtypically do not react with biological nucleophiles (although strainedalkynes may be susceptible to thiol attack). (3) The click chemistry canbe performed on living cells and whole organisms.

In some embodiments, the compounds of the present invention are used tomonitor azido-labeled molecules (e.g., azido sugar, protein bearingazido amino acids, lipids and site-specifically labeled proteins) inlive cells. The metabolic precursor peracetylatedN-azidoacetylmannosamine (Ac₄ManNAz) is metabolically adsorbed intocells of interest and incorporated into biomolecules that are expressedon the surface of the cells. In certain instances, the azido-sugarlabeled cells are exposed to a cyclooctyne (strained alkyne) reagentconjugated to a reporter group (e.g., dye), which generates a Huisgencycloaddition reaction. The cyclooctyne reagent can be added to cellculture medium and incubated with azido-sugar labeled cells underconditions that promote the click reaction. If the azido-sugar labeledcells are in a live organism, the cycloocyne reagent can be administeredto the organism by methods such as, but not limited to oral, topical andtransmembrane administration, and injection. As a result, thecyclooctyne-reporter conjugate covalently binds to the azido-sugarlabeled cells, which then labels the cells with the reporter. In otherinstances, the azido-sugar labeled cells are exposed to a phosphinereagent conjugated to a reporter group (e.g., dye), which generates aStaudinger ligation between the phosphine and the azido sugar. Thiscovalently binds the phosphine-reporter conjugate to the azido-sugarlabeled cells, which are now detectable using commercially availableimaging systems.

In other embodiments, Ac₄ManNAz is administered to a whole organism. Incertain instances, it is injected into an animal (e.g., zebrafish,rodents, rabbits, dogs, sheep, goats, pigs, monkey, and humans). Thismethod delivers azides to cell surface sialoglycoconjugates on cellsfound in serum and various tissues, such as, but not limited to heart,spleen, liver, kidney, intestines and muscle. In some instances, aphosphine- or alkyne-reporter can be injected into the same animal togenerate a Staudinger ligation or Huisgen cycloaddition reaction,respectively, in vivo. The labeled tissues and cells can be monitoredand analyzed using whole animal imaging systems. In other instances,tissues or cells are extracted from an Ac₄ManNAz injected animal, andthen they are treated with a phosphine- or alkyne-reporter in vitro.Methods known to those skilled in the art, such as, but not limited toWestern blotting, ELISA, immunocytochemistry, mass spectrometry, andhigh-performance liquid chromatography can then be used to detectlabeled biomolecules.

Example 92

Example 92 illustrates methods of using compounds of the presentinvention to label biomolecules (e.g., proteins, lipids, carbohydrates,nucleic acids, amino acids, glycerol, fatty acids, and nucleotides) oncells as shown in Scheme 2. It also illustrates methods of in vitro andin vivo analysis of the labeled biomolecules that can serve as detectionprobes. The compounds can be applied to methodologies used toinvestigate disease and therapeutic development, such as but not limitedto tumor imaging, glycan labeling, in vivo imaging, and cell surfacemodification.

Example 93 illustrates an ELISA using click chemistry.

In this example, the reaction is carried-out with IRDye 800CW as thedye. A skilled artisan would appreciate that compounds of the presentinvention (e.g., compound 10) can be substituted for IRDye 800CW.

Metabolic Labeling of an Bioorthogonal Functional Group on Biomolecules

In some embodiments, an azido-labeled biomolecule is made using methodsknown to those skilled in the art. In certain instances, an unnaturalazido sugar is commercially available from a supplier (e.g.,Sigma-Aldrich). In other instances, the unnatural azido sugar, such asthe metabolic precursor peracetylated N-azidoacetylmannosamine(Ac₄ManNAz)) is synthesized according to methods known to those skilledin the art (see, e.g., Laughlin et al., Methods Enzymol, 415:230-250(2006)). To incorporate an azido sugar into biomolecules expressed oncells cultured in vitro, the modified sugar is added to the cell culturemedia and incubated with the cells (see, e.g., Bussink et al., J. LipidRes., 48: 1417-1421 (2007); Prescher et al., Nature, 430: 873-877(2004)). Typically, Ac₄ManNAz is added to a cell culture at a finalconcentration of about 50 μM and incubated for about 3 days in cellculturing conditions. To label biomolecules expressed on cells in anorganism, an azido sugar (e.g., Ac₄ManNAz) is administered in a solutionto the organism by injection (e.g., intraperitoneal injection) at anappropriate injection schedule to ensure optimal incorporation andexpression of the modified biomolecule. See Chang et al., Proc. Natl.Acad. Sci U.S.A., 107:1821-1826 (2010). Non-limiting examples oforganisms include fish, rodents, rabbits, dogs, sheep, goats, pigs,monkey, and humans. Typically, Ac₄ManNAz is injected intraperitoneallyat a dose of 300 mg/kg in DMSO solution into mice once daily for 7 days.

Generation of PHOS-FLAG-Reporter Probe

Scheme 2 illustrates one embodiment of the invention, wherein aPHOS-FLAG-800CW probe is used to label specific azido-sugar labeledbiomolecules in living cells and organisms. In some embodiments, aphosphine-FLAG peptide conjugate (PHOS-FLAG; described in Laughlin etal., Methods Enzymol, 415:230-250 (2006)) is coupled to a reporter group(e.g., dye) using methods known to those skilled in the art. In certaininstances, the phosphine-FLAG peptide conjugate is labeled with IRDye800CW NHS ester (LI-COR) according to the manufacturer's instructions.The resulting PHOS-FLAG-800CW probe can then be covalently linked via aStaudinger ligation to an azido-labeled biomolecule expressed by a cell.

Detection of Biomolecules Labeled by Copper-Free Click Chemistry

Scheme 2 also illustrates a method of labeling a modified biomoleculefound on cell with a PHOS-FLAG-Reporter probe using “click” chemistry.

In some embodiments, a PHOS-FLAG-800CW probe is injected into an animalhaving cells that express azido-labeled biomolecules. In particularinstances, mice are injected intraperitoneally once with PHOS-FLAG-800CW(0.16 mmol/kg). Labeling of the cells with the near-infrared dye isdetected using a whole animal detection system (e.g., Pearl Imager(LI-COR)). Dye-labeled biomolecules, tissues and cells from the animalcan be harvested from a euthanized animal and analyzed using imagers(e.g., Odyssey System (LI-COR)).

In other embodiments, a PHOS-FLAG-800CW probe is added to an in vitrocell culture and incubated in conditions that promote a Staudingerligation reaction between the phosphine of the probe and the azide ofthe biomolecule. In certain instances, the “click” reaction is performedaccording to the following steps: 1) azido-labeled cells are collected;2) they are centrifuged at 1,500 rpm for 10 minutes, 3) they are washedthree times in cold PBS, 4) they are resuspended in 2% (v/v) fetal calfserum in PBS, 5) they are incubated with about 0.5 mM PHOS-FLAG-800CWprobe at room temperature for 3 hours under mild shaking, 6) the cellsare collected by centrifugation, and 7) they are washed three times withcold PBS. As a result of click chemistry, the labeled biomolecule iscovalently linked to a FLAG tag and near-infrared dye via the clickproduct. In some instances, the near-infrared dye-labeled cells orbiomolecules are detected and analyzed using a detection system (e.g.,Odyssey System (LI-COR)).

In certain embodiments, an ELISA-type assay is performed to detect theFLAG-tagged biomolecules expressed on cells. Protocols for ELISA-typeassays and immunocytochemistry are known to those of skill and describedin detail in reference books such as, Antibodies: A Laboratory Manual(ed. Harlow and Lane), Cold Spring Harbor Laboratory Press, New York,1988; Methods in Molecular Biology, Volume 42: ELISA, Theory andPractice (ed. Coligan et al.), Humana Press, New Jersey, 1995; andImmunoassay (ed. Diamandis and Christopoulos), Academic Press, New York,1996. In some aspects, the labeled cells are incubated with ahorseradish peroxidase (HRP)-conjugated anti-FLAG antibody at conditionsoptimal for antibody binding. HRP conjugated anti-FLAG antibodies arecommercially available from suppliers such as, but not limited to,Sigma-Aldrich (St. Louis, Mo.), Cell Signaling (Danvers, Mass.), ProteinMods (Madison, Wis.), Prospec Bio (East Brunswick, N.J.). Theantibody-labeled cells are exposed to a luminol substrate that isoxidized by HRP in a chemiluminescent reaction. The light-emittingreaction is detectable using imaging systems such as, but not limited toOdyssey Fc System (LI-COR). In other aspects, the FLAG taggedbiomolecules are extracted from the cells using methods known to thoseskilled in the art. Descriptions of methods for the isolation ofbiomolecules and the detection of FLAG-tagged biomolecules can be foundin references such as, but not limited to, Current Protocols inMolecular Biology (ed. Ausubel et al.), Wiley, New Jersey, 2011; andCurrent Protocols in Immunology (ed. Coligan et al.), Wiley, New Jersey,2011.

Example 94

Example 94 illustrates the compounds of the present invention withtechnology similar to Rutjes (cf. ChemBioChem 2007, 8, 1504-1508) usingcopper-free click chemistry reaction conditions as shown in Scheme 3.This example illustrates a method of covalently binding a near-infrareddye to selectively modified biomolecules that are expressed by cells.Non-limiting examples of applications of the methods described here inare tumor imaging, glycan labeling, in vivo labeling, cell surfacemodification, The method is based on a tandem [3+2]cycloaddition-retro-Diels-Alder ligation method that results in a stable1,2,3-triazole linkage.

In this example, the reaction is carried-out with IRDye 800CW as thedye.

Scheme 3 illustrates a method of linking a near-infrared dye to anazido-labeled biomolecule. In some embodiments, firstly, anoxanorbornadiene is coupled to a near-infrared dye (e.g., IRDye 800CW)via an amidation reaction to generate an IRDye800CW-oxanorbornadiene.Next, the IRDye800CW-oxanorbornadiene reagent is incubated with cellsexpressing an azido labeled biomolecule, thereby creating a “click”reaction. Typically, azido labeled cells are generated following methodsdescribed in Example 112. The tandem [3+2] cycloaddition andretro-Diels-Alder reactions generate a furan molecule and a triazolelinkage between the biomolecule and the dye, thereby labeling targetedcells with a dye.

In certain embodiments, a IRDye800CW-oxanorbornadiene reacts with aselectively modified azido-biomolecule on cells in an animal. TheIRDye800CW-oxanorbornadiene can be administered (e.g., injection, oral,transdermal and topical) to an animal. In some instances, theIRDye800CW-labeled cells and biomolecules are monitored in the animalusing an infrared detection system (e.g., Pearl Imager). In otherinstances, cells and tissues from the animal are harvested and analyzedusing techniques known to those in skilled in the art, such as, but notlimited to ELISA, FLISA, Western, histology, immunocytochemistry, andimaging. Methods including protocols are available in references suchas, but not limited to Current Protocols in Molecular Biology (ed.Ausubel et al.), Wiley, New Jersey, 2011; Current Protocols in ProteinScience (ed. Coligan et al.), Wiley, New Jersey, 2011; and CurrentProtocols in Immunology (ed. Coligan et al.), Wiley, New Jersey, 2011.In some instances, the labeled biomolecules and cells are monitoredusing techniques described herein.

In certain embodiments, the labeled biomolecule is detected andidentified using methods such as, but not limited to high-performanceliquid chromatography (HPLC) and liquid chromatography-mass spectrometry(LC-MS). Methods of detecting dye-labeled cells and biomolecules aredescribed in references, for example, Peptide Characterization andApplication Protocols (ed. Fields), Humana Press, New Jersey, 2007;Sample Preparation in Biological Mass Spectrometry (ed. Ivanov andLazarev), Springer, New York, 2010; and Proteomic Biology Using LC-MS:Large Scale Analysis of Cellular Dynamics and Function, (ed. Takahashiand Isobe), Wiley, New Jersey, 2008. In some instances, the labeledbiomolecule is a constituent of a molecular complex and methods ofdissociating, separating or modifying the complex are used prior toperforming methods for detecting and identifying the individual labeledpeptides. Examples include, but are not limited to LC-MS with peptidemass fingerprinting and tandem MS (LC-MS/MS).

Example 95

Example 95 illustrates the synthesis of a fluorescence-quenching dyesodium6-((E)-2-((E)-2-(3-((E)-2-(5-(bis(3-sulfonatopropyl)amino)-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indolium-2-yl)vinyl)-2-(2-fluorophenyecyclohex-2-enylidene)ethylidene)-1,1-dimethyl-7-sulfonato-1H-benzo[e]indol-3(2H)-yl)hexanoate(62).

Compound 62 was prepared by combining 50 mg of IRDye® QC-1 Carboxylate(63, LI-COR Biosciences), 9.8 mg of 2-fluorophenylboronic acid, 4.0 mgof Pd(PPh₃)₄ 16.4 mg of sodium acetate, 200 μL of 2-methoxyethanol, and2 mL of water. The mixture was heated at reflux for 1.5 hours under anitrogen atmosphere. The compound was purified by reverse-phase C18chromatography using acetonitrile/water yielding 50 mg of product.Absorbance: λ_(Water)=785 nm, λ_(MeOH)=777 nm.

Example 96

Example 96 illustrates the synthesis of sodium3,3′-(2-((E)-2-((E)-3-((E)-2-(3-(6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl)-1,1-dimethyl-7-sulfonato-1H-benzo[e]indol-2(3H)-ylidene)ethylidene)-2-(2-fluorophenyl)cyclohex-1-enyl)vinyl)-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indolium-5-ylazanediyl)dipropane-1-sulfonate(64).

Compound 64 was prepared by combining 60 mg of compound 62, 32 mg ofN,N′-disuccinimidyl carbonate, 10.8 μL of N,N-diisopropylethylamine, and3 mL of DMSO. The mixture was stirred at room temperature for 30minutes, precipitated into diethyl ether, and then purified byreverse-phase C18 chromatography using acetonitrile/water.

Example 97

Example 97 illustrates the synthesis of a fluorescence-quenching dyesodium1-(6-(6-aminohexylamino)-6-oxohexyl)-2-((E)-2-((E)-3-((E)-2-(3,3-dimethyl-5-sulfonato-1-(4-sulfonatobutyl)indolin-2-ylidene)ethylidene)-2-(4-sulfonatophenoxy)cyclohex-1-enyl)vinyl)-3,3-dimethyl-3H-indolium-5-sulfonate(800CW-Hexamethylenediamine, 65).

Example 98

Example 98 illustrates the synthesis of a strainedcycloalkyne-containing Compound 79 derivative for click chemistry(Compound 79-DBCO, 66).

To a solution of Compound 64 (Example 96, 0.8 mg, 6.1×10⁻⁴ mmol) andN,N-diisopropylethylamine (0.001 mL, 5.7×10-3 mmol) in anhydrousdimethylsulfoxide (0.8 mL) was added DBCO-Amine (from Jena Bioscience,0.5 mg, 1.8×10-3 mmol) in one portion. The reaction was allowed toproceed at ambient temperature for 2 h, with periodic agitation at15-min intervals. After HPLC analysis indicated complete consumption ofCompound 64, the crude product was precipitated in anhydrous diethylether (10 mL). The ethereal supernatant was decanted and the precipitatewas purified by prep-HPLC to afford the desired product Compound 87 as ateal solid (0.8 mg, 90%). UV/Vis (acetonitrile/water=1:1) λmax=790 nm;LRMS (ES/water), m/z calculated for C73H81FN5O14S4 [M+H]+ 1398.46. found1398.5.

Example 99

Example 99 illustrates the synthesis of an azide-containing Compound 62derivative for click chemistry (Compound 79-PEG-Azide, 67).

To a solution of Compound 64 (Example 96, 0.8 mg, 6.1×10-4 mmol) andN,N-diisopropylethylamine (0.001 mL, 2.3×10-3 mmol) in anhydrousdimethylsulfoxide (0.8 mL) was added11-azido-3,6,9-trioxa-undecan-1-amine (0.5 mg, 1.8×10-3 mmol) in oneportion. The reaction was allowed to proceed at ambient temperature for2 h, with periodic agitation at 15-min intervals. After HPLC analysisindicated complete consumption of Compound 81, the crude product wasprecipitated in anhydrous diethyl ether (10 mL). The etherealsupernatant was decanted and the precipitate was purified by prep-HPLCto afford the desired product Compound 67 as a teal solid (0.7 mg, 81%).UV/Vis (acetonitrile/water=1:1) λmax=790 nm; LRMS (ES/water), m/zcalculated for C63H83FN7O16S4 [M+H]+ 1340.48. found 1340.5 and 671.0[M+2H]2+.

Example 100

Example 100 illustrates the click chemistry synthesis of Compound10/Compound 62 conjugate (Compound 10-Compound 62 Click Product, 68).

To a solution of azide 67 (150 μg, 1.1×10⁻¹ mmol) in water (150 μL) wasadded a solution of Compound 10-PEG-Phosphine (59) (92 μg, 6.3×10⁻²μmol) in water (50 μL). The reaction was allowed to proceed at ambienttemperature for 1 hour, then maintained at 40° C. for 3 hours. AfterHPLC analysis showed complete consumption of Compound 10-PEG-Phosphine,the reaction mixture was filtered and directly purified by HPLC.Fractions containing the presumed Compound 10-Compound 79 Click Product68 were combined and concentrated in vacuo. The exact yield was notdetermined. UV/Vis (acetonitrile/water=1:1) λ_(max) 1=778 nm,λ_(max)2=677 nm; LRMS (ES/water), m/z calculated for C₁₂₃H₁₅₀ClN₉O₃₃PS₇[M+H]⁺ 2570.8. found 858.5 [M+3H]³⁺.

Example 101

Example 101 illustrates the synthesis of the fluorescence-quenching dyesodium(E)-2-((E)-2-(3-((E)-2-(5-(bis(3-sulfonatopropyl)amino)-3,3-dimethyl-1-(3-sulfonatopropyl)-3H-indolium-2-yl)vinyl)-5-carboxy-2-(2-fluorophenyl)cyclohex-2-enylidene)ethylidene)-1,1-dimethyl-3-(3-sulfonatopropyl)-2,3-dihydro-1H-benzo[e]indole-6,8-disulfonate(69).

Compound 69 was prepared by combining 200 mg of Bromo Dye 70, 31 mg of2-fluorophenylboronic acid, 12.7 mg of Pd(PPh₃)₄, 36.1 mg of sodiumacetate, 800 μL of 2-methoxyethanol, and 8 mL of water. The mixture washeated at reflux for 45 minutes under a nitrogen atmosphere. To thereaction was then added 1 mL of 10% sulfuric acid solution and refluxwas continued for 90 minutes. The compound was purified by reverse-phaseC18 chromatography using acetonitrile/water yielding 130 mg of product.Absorbance: λ_(Water)=779 nm.

Example 102

Example 102 illustrates the synthesis of an NHS ester of Compound 93(71).

Compound 71 was prepared by combining 130 mg of compound 69, 72 mg ofN,N′-disuccinimidyl carbonate, 25 μL of N,N-diisopropylethylamine, and 8mL of DMSO. The mixture was sonicated at room temperature for 120minutes, precipitated into diethyl ether, and then purified byreverse-phase C18 chromatography using acetonitrile/water yielding 80 mgof product.

Example 103

Example 103 illustrates the synthesis of a strainedcycloalkyne-containing Compound 69 derivative for click chemistry (72).

This compound was prepared in a manner similar to that used for 66(Example 120) from the commercially available DBCO-Amine. The exactyield was not determined. UV/Vis (acetonitrile/water=1:1) λ_(max)=799nm; LRMS (ES/water), m/z calculated for C₇₁H₇₇FN₅O₂₀S₆ [M+H]⁺ 1530.34.found 765.9 [M+2H]²⁺.

Example 104

Example 104 illustrates the synthesis of an azide-containing Compound 69derivative for click chemistry (Compound 69-PEG-Azide, 73).

This compound was prepared in a manner similar to that used for 67(Example 99) from commercially available11-azido-3,6,9-trioxaundecan-1-amine. The exact yield was notdetermined. UV/Vis (acetonitrile/water=1:1) λ_(max)=799 nm; LRMS(ES/water), m/z calculated for C₆₁H₇₉FN₇O₂₂S₆ [M+H]⁺ 1472.35. found736.9 [M+2H]²⁺.

Example 105

Example 105 illustrates the synthesis of an azide-containing Compound 10derivative for click chemistry (Compound 10-PEG-Azide, 74).

This compound was prepared in a manner similar to that used for 67(Example 99) from the commercially available11-azido-3,6,9-trioxaundecan-1-amine. The exact yield was notdetermined. UV/Vis (acetonitrile/water=1:1) λ_(max)=676 nm; LRMS(ES/water), m/z calculated for C₄₇H₆₁ClN₇O₁₃S₃ [M+H]⁺ 1062.31. found1060.6 [M−H]⁻, 1082.6 [M+Na−2H]²⁻.

Example 106

Example 106 illustrates the synthesis of an strainedcycloalkyne-containing Compound 10 derivative for click chemistry(Compound 10-DBCO, 75).

This compound was prepared in a manner similar to that used for compound66 (Example 98) from the commercially available DBCO-Amine. The exactyield was not determined. UV/Vis (acetonitrile/water=1:1) λ_(max)=676nm; UV/Vis (acetonitrile/water=1:1) λ_(max)=676 nm; LRMS (ES/water), m/zcalculated for C₅₇H₅₉ClN₅O₁₁S₃ [M+H]⁺ 1120.30. found 1120.6. 560.9[M+2H]²⁺.

Example 107

Example 107 illustrates the synthesis of Compound 10/Compound 69conjugate (Compound 10/Compound 69 Click Product 1; 76).

A solution of Compound 72 (0.165 mg, 1.0×10⁻⁴ mmol) in water (0.150 mL)was mixed with a solution of Compound 74 (0.055 mg, 5.0×10⁻⁵ mmol) inwater (0.050 mL). The reaction mixture was agitated for 30 seconds andthen allowed to proceed at ambient temperature for 2 hours. After HPLCanalysis showed the complete consumption of Compound 74, the reactionmixture was directly purified by reverse-phase HPLC to afford theproduct Compound 76 as a blue solid that was a mixture of the twotriazole cycloaddition regioisomers. The exact yield was not determined.UV/Vis (acetonitrile/water=1:1) λ_(max)1=679 nm, λ_(max)2=799 nm; LRMS(ES/water), m/z calculated for C₁₁₈H₁₃₇ClF₁₂O₃₃S₉ [M+H]⁺ 2591.65. found1295.0 [M−2H]²⁻, 863.1 [M−3H]³⁻.

Example 108

Example 108 illustrates the synthesis of another Compound 10/Compound 69conjugate (Compound 10/Compound 69 Click Product 2; 77).

A solution of Compound 75 (0.060 mg, 5.0×10⁻⁵ mmol) in water (0.050 mL)was mixed with a solution of Compound 73 (0.160 mg, 1.0×10⁻⁴ mmol) inwater (0.150 mL). The reaction mixture was agitated for 30 seconds andthen allowed to proceed at ambient temperature for 2 hours. After HPLCanalysis showed the complete consumption of Compound 73, the reactionmixture was directly purified by reverse-phase HPLC to afford theproduct Compound 77 as a blue solid that was a mixture of the twotriazole cycloaddition regioisomers. The exact yield was not determined.UV/Vis (acetonitrile/water=1:1) λ_(max)1=679 nm, λ_(max)2=799 nm; LRMS(ES/water), m/z calculated for C₁₁₈H₁₃₇ClFN₁₂O₃₃S₉ [M+H]⁺ 2591.65. found1295.6 [M−2H]²⁻

Example 109 Comparison of Dye Emission Maxima and Quenching

Solutions of dye-linker standards and dye-linker-quencher samples werediluted into PBS buffer (pH 7.4) to give a dye-specific absorbance lessthan 0.2 AU. The fluorescence spectra of each dilution were then takenat a consistent excitation wavelength (670 nm for Compound 10 and 770 nmfor 800CW). The emission spectra were collected from 680-1000 nm for theCompound 10 samples and 780-1000 nm for the 800CW samples.

Quencher Example Emission Percent Version Sample Name Number MaximumQuenching Compound Compound 10-PHOS- Example 89 3.02 × 10⁶ 62 OX (ref)(60) Compound 10- Example 100 1.05 × 10⁶ 65.1 Compound 62-Click 1 (68)Compound Compound 10-PHOS- Example 89 3.02 × 10⁶ 69 OX (ref) (60)Compound 10- Example 107 2.78 × 10⁵ 90.8 Compound 69-Click 1 (76)Compound 10- Example 108 4.99 × 10⁵ 83.5 Compound 69.-Click 2 (77)

Example 110 In Vivo Use of Evaluation of Compound 10

In vitro data was obtained to provide sufficient data on the relativenon-specific binding of the dye which included tests with 10/RGD and10/EGF conjugates, two targeted imaging agents. In vivo evaluation ofthe carboxylate 10 (non-reactive form) and a conjugated form targeted toEGFR, 10/EGF, were used to assess effective use in a working model onthe Pearl Impulse Imaging System.

Procedure:

Compound 10/RGD and 10/EGF were prepared to be used in conjunction with10 to estimate relative non-specific binding of the dye relative to alabeled targeted agent. The on-cell Western was the format used andimaged on the Odyssey Sa.

Nude mice were used for the clearance study with the carboxylate and forthe targeted agent evaluation. A dose of 2 nmol 10 was administeredintravenously (IV) via the tail vein of the mouse and serial imaged overthe next 48 h with the Pearl Impulse imaging system. Excised organs wereevaluated to assess organs with the highest retention.

Results:

In a plate-based evaluation of 10/RGD on U87GM cells, 10 was added toassess level of cell binding (FIG. 27). The results show low levels ofcell binding <0.5 μM for the carboxylate in comparison to a specifictargeted probe.

The initial batch was then tested in vivo to assess non-specificretention and clearance over a 44 hr period. The results showed that theprobe clears via the liver/gall bladder/intestinal tract and very littleretention in the kidneys, if at all.

The Total Signal Intensity for each image was captured in a large ROIand plotted (FIG. 28). The carboxylate signal is dramatically reduced by4 hours with the largest contribution of signal being located in theintestinal tract. The spike at about 1 hour reflects the movement of theprobe to the periphery where it is closer to the surface of the skin andmore easily detected. An examination of excised organs (i.e., liver,heart, kidney, spleen, intestine, muscle, tumor) show very littleretention of probe after 44 hr post injection.

A test dose of 2 nmol of 10 was administered to a nude mouse (FIG. 29).This test dose was selected for this evaluation because it is theoptimal dose for two of the three probes (i.e., 10, 10/RGD, 10/EGF).

10/EGF Targeted Agent Testing:

Initial cell-based assays show the 10/EGF effectively binds to A431cells (high EGFR). Results are shown in FIG. 30 where increasingconcentrations of 10/EGF showed a dose dependent increase in binding. Acompetition assay confirms specificity to the EGFR. Results again showincreasing concentrations of unlabeled EGF effectively blocked thebinding of the labeled EGF.

In Vivo Analysis:

Testing for optimal dose in nude mice bearing A431 subcutaneous tumorswas done. Four doses were tested: 0.5, 1, 2, and 3 nmoles. Two mice wereused per treatment dosage. A431 tumor cells were implanted on the righthip and allowed to reach approximately 0.5 cm. FIG. 31 presents dorsalviews of all animals under a common LUT.

Results of the dose testing suggest an effective dose for 10/EGF shouldbe between 2-3 nmol per injection. A slightly higher level of backgroundcan be expected as the dose increases, but the tumor was visible fordoses of 1-3 nmol.

Excised organs (48 h post injection) from mice in the dose study showthat the level of signal retained in the tumor is highest for the doses1-3 nmol. These doses produced much brighter signal from the tumorcells, especially when compared to 10 itself, which had very littlesignal from the tumor cells.

The disclosures of all articles and references cited in thisapplication, including patent applications [e.g., U.S. PatentApplication No. 61/170,579 (filed Apr. 17, 2009), 61/184,750 (filed Jun.5, 2009), Ser. No. 12/820,077 (filed Jun. 17, 2010)], patents, PCTpublications (e.g., W.I.P.O. Application No. PCT/US2010/031434, filedApr. 16, 2010), and non-patent publications, are incorporated herein byreference for all purposes. This application also incorporates byreference the full text of the application “Fluorescent Imaging withSubstituted Cyanine Dyes,” Attorney Docket Number 85409-820353(020031-010610PC), which also claims priority to U.S. Provisional PatentApplication Nos. 61/405,158 and 61/405,161.

References regarding click chemistry are shown below and are herebyincorporated by reference.

-   1. Ghosh, A. K.; Duong, T. T.; McKee, S. P.; Thompson, W. J.    “NN′-disuccinimidyl carbonate: a useful reagent for    alkoxycarbonylation of amines.” Tetrahedron Lett. 1992, 33,    2781-2784.-   2. Ghosh, A. K.; McKee, S. P.; Duong, T. T.; Thompson, W. J. “An    efficient synthesis of functionalized urethanes from azides.” Chem.    Commun. 1992, 1308-13010.-   3. Højfeldt, J. W.; Blakskjær, P.; Gothelf, K. V. “A Cleavable    Amino-Thiol Linker for Reversible Linking of Amines to DNA.” J. Org.    Chem. 2006, 71, 9556-9559.-   4. Bertozzi, C. R.; Bednarski, M. D. “The synthesis of    heterobifunctional linkers for the conjugation of ligands to    molecular probes.” J. Org. Chem. 1991, 56, 4326-4329.-   5. Schwabacher, A. W.; Lane, J. W.; Scheisher, M. W.; Leigh, K. M.;    Johnson, C. W. “Desymmetrization Reactions: Efficient Preparation of    Unsymmetrically Substituted Linker Molecules.” J. Org. Chem. 1998,    63, 1727-1729.-   6. Website: http://www.baseclick.eu and references therein.-   7. Chan, T. R.; Higraf, R.; Sharpless, K. B.; Fokin, V. V.    “Polytriazoles as Copper(I)-Stabilizing Ligands in Catalysis.” Org.    Lett. 2004, 6, 2853-2855.-   8. El-Sagheer, A. H.; Brown, T. “Click Chemistry with DNA.” Chem.    Soc. Rev. 2010, 39, 1388-1405.-   9. C. W. Tornoe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67,    3057-3064; V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B.    Sharpless, Angew. Chem. 2002, 114, 2708-2711; Angew. Chem. Int. Ed.    2002, 41, 2596-2599.-   10. C. J. Burrows, J. G. Muller, Chem. Rev. 1998, 98, 1109-1151.-   11. T. R. Chan, R. Hilgraf, K. B. Sharpless, V. V. Fokin, Org. Lett.    2004, 6, 2853-2855.-   12. J. Gierlich, G. A. Burley, P. M. E. Gramlich, D. M. Hammond, T.    Carell, Org. Lett. 2006, 8, 3639-3642. F. Seela, V. R. Sirivolu,    Chem. Biodiversity 2006, 3, 509-514.-   13. P. M. E. Gramlich, S. Warncke, J. Gierlich, T. Carell, Angew.    Chem. 2008, 120, 3491-3493; Angew. Chem. Int. Ed. 2008, 47,    3442-3444.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication, patent or patent application were specifically andindividually indicated to be incorporated by reference. Although theforegoing invention has been described in some detail by way ofillustration and example for purposes of clarity of understanding, itwill be readily apparent to those of ordinary skill in the art that, inlight of the teachings of this application, that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

What is claimed is:
 1. A bioconjugate compound of Formula II:

wherein Q^(L) is a portion of a polymethine bridge selected from thegroup consisting of:

wherein Q^(L) is the central portion of either a five- or aseven-polymethine-carbon polymethine bridge; each R¹ is a memberindependently selected from the group consisting of -L-Y—Z and alkylthat is additionally substituted with from 0 to 1 R¹³ and from 0 to 1R^(L); wherein the alkyl is optionally interrupted by at least oneheteroatom; each R^(2a) and R^(2b) is a member independently selectedfrom the group consisting of alkyl, alkenyl, hydroxyalkyl, alkoxyalkyl,aminoalkyl, amidoalkyl, alkylthioalkyl, carboxyalkyl,alkoxycarbonylalkyl, and sulfonatoalkyl; wherein a carbon of the memberis additionally substituted with from 0 to 1 R^(L); or, alternatively, aR^(2a) and R^(2b) pair, together with the ring carbon to which theR^(2a) and R^(2b) are bonded, join to form a spirocycloalkyl ring,wherein the spirocycloalkyl ring is additionally substituted with from 0to 6 R¹⁴ and from 0 to 1 R^(L), or an exocyclic alkene, wherein theexocyclic alkene is additionally substituted with from 0 to 2 R¹⁴ andfrom 0 to 1 R^(L); each R³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), andR^(6b) is a member independently selected from the group consisting ofhydrogen, alkyl, alkenyl, halo, hydroxyl, alkoxy, amino, cyano,carboxyl, alkoxycarbonyl, amido, sulfonato, alkoxyalkyl, carboxyalkyl,alkoxycarbonylalkyl, and sulfonatoalkyl; wherein a carbon of the memberis additionally substituted with from 0 to 1 R^(L); or, alternatively, apair of said members that is selected from the group consisting of R³and R^(4a), an R^(4a) and R^(5a), and an R^(5a) and R^(6a), togetherwith the pair of atoms to which the pair of said members is bonded,joins to form an aryl ring, wherein the aryl ring is additionallysubstituted with from 0 to 2 R¹⁴ and from 0 to 1 R^(L); each R⁷ is amember independently selected from the group consisting of hydrogen andalkyl; wherein the alkyl is additionally substituted with from 0 to 3R¹⁴ and from 0 to 1 R^(L); or, alternatively, both R⁷, together with theintervening segment of the polyene to which both R⁷ are bonded, join toform a ring, wherein said ring is selected from the group consisting ofa cycloalkyl and a heterocyclyl ring; wherein the ring is additionallysubstituted with from 0 to 3 R¹⁴ and from 0 to 1 R^(L); R⁸, R⁹, R¹⁰,R¹¹, and R¹² are each a member independently selected from the groupconsisting of hydrogen, alkyl, alkenyl, halo, alkoxy, sulfonato,hydroxyl, amino, carboxyl, alkoxycarbonyl, cyano, amido, thioacetyl, and-L-Y—Z; wherein, if present, at least one member selected from the groupconsisting of R⁸, R⁹, and R¹⁰ is -L-Y—Z; each R¹³ is a memberindependently selected from the group consisting of hydroxyl, amino,carboxyl, alkoxycarbonyl, cyano, amido, sulfonato, and thioacetyl; eachR¹⁴ is a member independently selected from the group consisting ofalkyl, alkenyl, halo, hydroxyl, alkoxy, amino, cyano, carboxyl,alkoxycarbonyl, amido, sulfonato, alkoxycarbonylalkyl, and alkoxyalkyl;wherein the alkyl or alkenyl is additionally substituted with from 0 to1 R¹³ and from 0 to 1 R^(L); each L is an optional member independentlyselected from the group consisting of a bond, a C₁-C₂₀ alkylene, and aC₁-C₂₀ alkenylene; wherein the alkylene or alkenylene is optionallyinterrupted by at least one heteroatom; each Y is an optional memberindependently selected from the group consisting of a bond, —O—, —S—,—NH—, —NHC(O)—, —C(O)NH—, —NR¹⁵—, —NR¹⁵C(O)—, —C(O)NR¹⁵—, —N(Z)—,—N(Z)C(O)—, and —C(O)N(Z)—; each Z is independently selected from thegroup consisting of -L-R¹³ and -L-R^(L); alternatively, —Y—Z is a memberselected from the group consisting of —N(Z)₂, —N(Z)C(O)Z, and—C(O)N(Z)₂, and the two Z groups may optionally be linked to form acycloalkynyl group; each R¹⁵ is a member independently selected from thegroup consisting of alkyl and alkoxycarbonylalkyl, wherein the alkyl isoptionally interrupted by at least one heteroatom; each R^(L) comprisesa linking group and a biomolecule connected thereby, wherein thecompound comprises at least one R^(L); wherein the biomolecule isselected from the group consisting of hyaluronan, a tetracyclinederivative, avidin, biotin, streptavidin, a peptide, and a protein; andwherein said compound has a balanced charge.
 2. The compound of claim 1,wherein Q is:

wherein each R¹ is an independently selected sulfonatoalkyl or anindependently selected alkyl that is additionally substituted with from0 to 1 R¹³; each R^(2a) and R^(2b) is a member independently selectedfrom the group consisting of alkyl, carboxyalkyl, and sulfonatoalkyl;each R³, R^(4a), R^(4b), R^(5a), R^(5b), R^(6a), and R^(6b) is a memberindependently selected from the group consisting of hydrogen, alkyl,carboxy, carboxyalkyl, halo, sulfonato, and sulfonatoalkyl; R⁸, R⁹, R¹⁰,R¹¹, and R¹² are each a member independently selected from the groupconsisting of hydrogen, alkyl, halo, sulfonato, sulfonatoalkyl, and-L-Y—Z; wherein at least one member selected from the group consistingof R⁸, R⁹, and R¹⁰ is -L-Y—Z; each R¹³ is a member independentlyselected from the group consisting of hydroxyl, amino, and carboxyl;each R¹⁴ is a member independently selected from the group consisting ofalkyl, alkenyl, carboxyl, alkoxycarbonyl, amido, alkoxycarbonylalkyl,and halo; each L is a member independently selected from the groupconsisting of a bond, a C₁-C₂₀ alkylene, and a C₁-C₂₀ alkenylene; andeach Y is a member independently selected from the group consisting of abond, —O—, —NHC(O)—, —C(O)NH—, —NR¹⁵C(O)—, —C(O)NR¹⁵—, —N(Z)—,—N(Z)C(O)—, and —C(O)N(Z)—; alternatively, —Y—Z is a member selectedfrom the group consisting of —N(Z)₂, —N(Z)C(O)Z, and —C(O)N(Z)₂, and thetwo Z groups may optionally be linked to form a cycloalkynyl group. 3.The compound of claim 2, having the formula:

wherein M is an alkali metal ion.
 4. The compound of claim 1, whereinthe bioconjugate is hyaluronan.
 5. The compound of claim 1, wherein thebioconjugate is selected from the group consisting of an avidin,streptavidin, and biotin.
 6. The compound of claim 1, wherein thebioconjugate is a tetracycline derivative.
 7. The compound of claim 1,wherein the bioconjugate is a peptide.
 8. The compound of claim 7,wherein the peptide is a cyclic RGD peptide or an acyclic RGD peptide.9. The compound of claim 1, wherein the bioconjugate is a protein. 10.The compound of claim 9, wherein the protein is EGF.
 11. The compound ofclaim 9, wherein the protein is an antibody or an antibody fragment. 12.The compound of claim 11, wherein the antibody or antibody fragment isan antibody selected from the group consisting of a goat anti-mouse(GAM) antibody and a goat anti-rabbit (GAR) antibody.
 13. The compoundof claim 3, wherein the bioconjugate is hyaluronan.
 14. The compound ofclaim 3, wherein the bioconjugate is selected from the group consistingof an avidin, streptavidin, and biotin.
 15. The compound of claim 3,wherein the bioconjugate is a tetracycline derivative.
 16. The compoundof claim 3, wherein the bioconjugate is a peptide.
 17. The compound ofclaim 16, wherein the peptide is a cyclic RGD peptide or an acyclic RGDpeptide.
 18. The compound of claim 3, wherein the bioconjugate is aprotein.
 19. The compound of claim 18, wherein the protein is EGF. 20.The compound of claim 18, wherein the protein is an antibody or anantibody fragment.
 21. The compound of claim 20, wherein the antibody orantibody fragment is an antibody selected from the group consisting of agoat anti-mouse (GAM) antibody and a goat anti-rabbit (GAR) antibody.